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:
Add 9ml of YPD to 3 test tubes:
D2O YPD
D2O YPD
DDW YPD
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
Incubate at 30C and shake
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.
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.
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:
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.
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:
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!
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.
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.
So I let the yeast from Friday incubate over the weekend, then there was a powerfailure. 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.
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.
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:
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.
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.
Incubate at 30C.
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.