Category Archives: Yeast

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

Basic Observations of the D2O Adapted Yeast compared to WT Yeast

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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, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 49

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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.

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

D2O adaptation day 48

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Results:

  • d2o yeast gen 37 – 3.079 at 24f
  • d2o yeast gen 38 – 0.422 at 0h
  • ddw yeast – 3.039 at 24h
  • ddw yeast – 0.312 at 0h

Sorry for the sloppiness, but I’m doing this via my phone.

Also yesterday I grew cells from the inoculating loop, and in 24h they grew to an absorbable of: 2.708 (d2o yeast) and 1.700 (ddw yeast).

I inoculated new samples by mixing 9ml of ypd with 1ml of the previous generation’s culture (from the inoculating loop cultures). I also inoculated yeast on solid media. There are 4 samples:

  • d2o yeast on d20 agarose ypd
  • d2o yeast on regular agar ypd
  • ddw yeast on d2o agarose ypd
  • ddw yeast on regular agar ypd

The intent is to compare colony growth on plates over time to see if there are differences between the environments.

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

D2O Adaptation Day 47

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So I let the yeast from Friday incubate over the weekend, then there was a power failure. Hopefully nothing major happened. Because of the power failure I’m postponing my morphology experiments for either tomorrow or Wednesday and also the D2O solid culture growth until tomorrow. In the mean time we have some data:

  • D2O yeast (generation 36) – 3.261 at 72h
  • D2O yeast (gen 37) – 1.019 at 0h

As per usual, 9ml of D2O YPD were mixed with 1ml of D2O yeast from generation 36. I also did 10ml of D2O YPD with a colony of yeast inoculated from the same sample (gen 36). Hopefully that batch minimizes the amount of cells affected by the power outage.

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

D2O Adaptation Day 44 Time Trials Results

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Via figshare:

Day 44 Yeast Adaptation Hourly Growth. Anthony Salvagno. figshare.
Retrieved 23:54, Nov 09, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.97284

So based on these results, it appears that there is a difference in growth rates between my adapted D2O yeast and yeast adapted to H2O and grown in D2O. The main evidence is that the absorbance of adapted yeast starts much lower than the nonadapted yeast, and after a couple hours surpasses the cell count of the nonadapted yeast. The nonadapted yeast had exhibited relatively little growth for the first 5 hours of the experiment, while the adapted yeast grew consistently over the duration of the experiment.

The other aspect of this experiment was to compare the growth of yeast in water after different incubation times to determine if the yeast is exhibiting log-phase growth. I would say it most definitely is. Both samples grow at about the same rate, just starting from different cell counts. This also means that the yeast inoculated in D2O was growing at this rate and was severely stressed when placed in the D2O medium.

The next experiments are to compare yeast grown in DDW YPD to yeast adapted and grown in D2O to yeast adapted to H2O and grown in D2O. Hopefully there will be some interesting results. And next from there will be to grow the adapted yeast in H2O to see if H2O causes similar stresses to D2O adapted yeast that D2O causes to H2O adapted yeast.

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

D2O Adaptation Day 44 Time Trial Setup

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Two days ago I inoculated a starter culture of yeast in DDW YPD, and let that grow for 48h. Yesterday, I inoculated another culture from the previously mentioned culture to ensure that my yeast for today’s experiment would be in the log-phase of growth. Then I took a sample of my normal D2O yeast and compared the growth of this yeast against all the others and made a new sample of the yeast grown in DDW YPD to be grown in D2O. This would be to finally determine if the growth of regular yeast in D2O is obviously different than the yeast that I’ve been incubating in D2O for the past 44 days. Here is my setup:

  1. Put 9ml of YPD in a test tube. I want 4 samples, with two samples being DDW YPD and the other two being D2O YPD.
  2. Add 1ml of culture to each sample. One sample has 24h incubated DDW YPD yeast, one has 48h incubated DDW YPD yeast (see above), a third has generation 36 D2O YPD yeast, and the final sample is 48h DDW YPD yeast in D2O.
  3. Incubate at 30C.
  4. Record absorbance via nanodrop hourly.

The main motivation (as I said above) is to compare the growth of my 44 day incubated D2O yeast vs regular yeast grown in D2O. Normally the yeast in D2O starts at a really low absorbance value for example ~0.020 compared to yeast in DDW (ie ~1.000) or D2O adapted (~0.700). I wanted to start with a much higher cell count to see if the growth would be any different.

The other motivation is to compare the growth of yeast after 24h of incubation vs 48h of incubation. I was worried that they time trials I’ve been doing haven’t been using yeast in log-phase growth, and if their growths are comparable I know that I’m not.

 

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

D2O Adaptation Day 44

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Results:

  • Yeast in DDW – 1.563 at 24h
  • Yeast in DDW – 3.054 at 48h
  • Yeast in D2O gen 35 – 3.351 at 24h
  • Yeast in D2O gen 36 – 0.742 at 0h

After 48h the yeast in DDW finally equaled the amount of yeast in D2O. I’m inferring nothing, but I’m just sayin’. Anyways I setup the 36th generation of D2O yeast with 9ml of D2O YPD and 1ml of generation 35 culture.

D2O adaptation, Paper Summaries, Water Type Effects on Organism Growth, Yeast

Effect of Hsp26 on Yeast Growth

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K. Unno, T. Kishido, and S. Okada, “Effect of over-expressed Hsp26 on cell growth in yeast,” Biol. Pharm Bull., vol. 21, pp. 631–633, 1998.

In an attempt to further understand the poorly written paper I summarized yesterday I’m going back to read the paper they cited pretty frequently. Hopefully this paper will answer some of the questions I had yesterday. Note time:

  • The motivation for this study is that there has been no observation of the effects of the over-expression or depression of hsp26. Apparently hsp26 is induced during the transition of log-phase growth to stationary phase in yeast growth.
  • As they mentioned in yesterday’s paper (this one is earlier by a few years), the yeast cells used were ssa1ssa2 and have a slower growth rate than the wt.
  • Like the methods from yesterday, they picked clones of ssa1ssa2. However, this time the clones they chose had different doubling times from each other. Yesterday they chose clones whose doubling times were comparable to the wt.
  • cells were incubated at 25C in a minimal dextrose medium (YPd? hahaha) supplemented with nutrients but lacking uracil. WHY???
  • They plot the doubling time of the clones of ssa1ssa2 and the wt. It didn’t occur to me that the growth rate of clones could be different. This may be interesting to explore for my D2O variant strain. It should be noted that the doubling time of the wt strain had little variance amongst clones. What is the mechanism for this?
  • Exactly what is the difference between clones of the wt and clones of ssa1ssa2???
  • they then relate the expression of hsp26 to doubling time and compare that to other proteins (hsp70, hsp90, hsp104, Ssb, and Kar2). They find that hsp26 is more closely correlated to doubling time than the other 3 hsp’s and make no claim about Ssb and Kar2. But they do state that the amounts of Ssb and Kar2 in the clones is similar to the wt. This is sort of true.

So based on subtle information in this paper, it seems the mutant ssa1ssa2 lacks the genes Ssa1 and Ssa2 which make the hsp70 enzyme. But this paper mentions that Ssa4 is inducible and also produces hsp70 when induced. So in yesterday’s paper when they said there was no hsp70 but then they got hsp70, I’m guessing they induced the Ssa4 gene to get some hsp70. Good thing they mentioned that!

In this paper the induced hsp70 levels are less than the wt, and their data shows that hsp70 levels increase with increased doubling time. So yeast that lack Ssa1 and Ssa2 but have Ssa4 have higher levels of hsp26 (than wt) and almost normal levels of hsp70 are at risk for longer growth rates (doubling time). So the proteins may be linked in the extreme case that the Ssa1 and 2 genes are off and the Ssa4 gene is on (both produce hsp70).

But how does this information relate to the paper yesterday? It doesn’t really, but it may explain why they were seemingly contradicting the amount of hsp70 they had in their cells.

Ok just quickly rereading the first few pages of yesterday’s paper and I still have no idea what is going on. No matter. Their results claim to show that some clone of ssa1ssa2, which is beyond arbitrary, is D2O tolerant. I’m not too sure that their methods apply to what I’m doing, since they are just looking for deuterium resistance and not necessarily an organism that is made of D instead of H. I will go over yesterday’s paper again later tonight/early tomorrow to make sure things still make no sense (but maybe I’ll have a moment of clarity?).

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

D2O Adaptation Day 43

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Results:

  • D2O Yeast gen 34 – 3.097 at 24h
  • DDW Yeast – 1.241 at 24h
  • D2O yeast gen 35 – 0.856 at 0h

Yesterday I did a starter culture and today it’s absorbance is 1.241 in 24 hours. I’m electing to continue incubation of that culture for another 24h and am starting a new culture in 10ml of DDW YPD inoculated via inoculation loop. I also continued the yeast growth of the D2O strain (9ml of D2O YPD and 1ml of culture from generation 34). All three samples are incubating at 30C in my shaker.