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).