Tag Archives: failed

Yeast Colonies in 60% D2O YPD

So I suck at my own microscopy technique of taking pictures and combining them. In each case I left out a pretty sizable portion of the colony. 🙁 Oh well. I’m going to do these experiments again (cause I made 2 sets of each type of solid media, 0%-100% D2O) and will be taking new images then. Can’t wait!!

Yeast morphology in D2O

Checking on my yeast to ensure there isn’t any bacterial growth, I noticed they look very different in different D2O concentrations. In 99% D2O the cells appear larger and more circular, while in 20% D2O the cells appear to be elongated. I’ll have to grow some cells in normal water for comparison.

Also I noticed that in 99% D2O the cells seemed to form small colonies of about 10-20 cells, while in 20% D2O I saw almost no evidence of colonial formation. I also saw no yeast tea party (no? NO? oh well…). I think the colonies aren’t so much clusters as they are chains of buds, because they all seem to be attached. I didn’t analyze very thoroughly though.

Yeast Adaptation Day 3

My original yeast is still going in D2O. There hasn’t been much growth in the past 48 hours. And to hopefully achieve a faster/more efficient form of adaptation, I’m growing yeast in slightly increasing amounts of D2O. Today I started growing in 20% D2O (mixed with DI water to save money).

After 48 hours of growth the yeast in DDW measured 3.327 in the nanodrop.

Also I noticed that the YPD in DDW aggregated. This took about 12 days. I’ve never had this happen in D2O, except one time it did in a 1ml amount in a cuvette. That took about 14 days. And it was next to a heat source during that entire time. So it would be interesting to test the aggregation affects of YPD in DI/DDW and D2O. If I can achieve an adapted form of yeast, growing in D2O YPD could be beneficial all around (cost effective).

D2O Adaptation Try 2: Day 1

Because I’m 90% confident that the adapted yeast was actually e. coli (based on images and smell) I’m going to restart the experiment. I started by cleaning the incubator/shaker. Might as well try and minimize e. coli outbreak…

Setup:

  • Add 9ml of D2O YPD (all that I had left) to test tube.
  • Inoculate a colony from yeast grown on D2O solid media.
  • Add 9ml of DDW YPD to test tube
  • Inoculate colony from yeast grown on D2O solid media.
  • Place in incubator at 30C and 185rpm

I also made a fresh batch of D2O YPD:

  • 4.6g of powder YPD
  • 92ml of D2O
  • stir until dissolved
  • filter (2um) into bottle

Individual cells grown in DDW and D2O

These images were acquired last week (Thursday evening).

It was discovered that individual cells of D2O adapted yeast are very rod like and potentially fissionable. This indicates either one of two things: (1) there has been contamination and this is either a fissionable yeast or bacteria, (2) D2O fucks shit up really messes with cells and these are really distressed. I’m inclined to believe it is contamination since I wasn’t personally overseeing the yeast propagation for almost 3 weeks.

So to check, (1)  I will regrow the yeast cells from the beginning with antibiotics, (2) grow a sample of this stuff with antibiotics, and (3) reintroduce these cells to DDW for a few days to see if the growth reverts back to wt yeast. Any of those experiments could reveal the truth, but I don’t think my yeast is antibiotic resistant so I’d have to figure out some way to achieve that.

The biggest issue is that money is getting tight and D2O and DDW is expensive, so I’ll need to develop some cost cutting methods.

Non-adapted yeast grown on D2O YPD Agar

The yeast colonies grown in D2O agar are finally big enough to compare to the other samples. It took almost a week to grow this much!

Up above is an image of a single colony, and another of a few colonies that have merged together. It seems that in the presence of D2O, the colonies grow quite smooth still, but a little asymmetrically. Since we know (from unpublished research) that D2O stabilizes microtubules, it would be interesting to compare these results with the morphology of colonies grown in taxol (a cancer drug known to stabilize microtubules).

D2O Adaptation Day 54

I’m forgoing future measurements. I have some data to post (in a few minutes) that may reveal potential contamination. And so to verify if the samples are contaminated or if a cool new phenomenon is occurring, I’m growing the D2O adapted yeast in DDW, to see if they revert back. Although, I don’t think that will reveal much either way.

In lieu of the daily measurements I’ll be taking microscope images of the cells as they grow.

Every day I will inoculate a few colonies from the previous generation (similar to what I have been doing, with an inoculating loop) into 10ml of fresh YPD (both D2O and DDW).

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!