Setup. Today I”m doing the time trial and I setup 3 samples, following my protocol:
- Yeast in DI YPD
- Yeast in 20% D2O YPD
- Yeast in 99% D2O YPD
Live Results:
Tag Archives: data
DI YPD Yeast Observation
I needed an image of wt yeast in DI YPD to compare with the morphology of later strains. As I ramp up the D2O concentration, I expect to see some differences and this is good information to have in general.
I don’t think there is much to infer from the 20% D2O comparison image, but if it’s worth anything there are a few cells in that image that are elongated, and I never saw that morphology in DI water.
E. coli cell morphology: From D2O Adaptation 1
Back in the day I was calling the bacteria grown here D2O adapted yeast. Boy was I stupid. Anyway, here is the data I took when trying to compare the morphologies. Despite my naivety, I think there can be interesting studies done with E. coli in D2O based on these experiments and the colony morphology experiments I did.
Also I’ve included actual yeast in D2O images, which show that yeast form “chain gangs.” Basically the buds never seal off and grow new buds and large clusters of bud chains grow. Hopefully I can analyze this more in the upcoming experiments.
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 2 Live Results
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).
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 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!
Microscope Pixel Calibration
It is important to provide scale when showing microscope images. Personally I’m not a huge fan of scale bars and would prefer to know the dimensions of an image and the conversion factor. For our camera/microscope at 10x magnification it’s almost exactly 1um/px. This calibration was performed by Andy Maloney using a 60x objective. See below for details.