Via figshare:
D2O Adaptation Day 9 – FAILED. Anthony Salvagno. figshare.
Retrieved 18:51, Oct 08, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.96344
There is a basic explanation of the experiment failure on the figshare page. See here for more thorough explanation.
So the Balloon Fiesta was this weekend and I didn’t get an opportunity to get in lab. But it didn’t much matter because the results I got Thursday and Friday of last week were not consistent/reliable. Out of that frustration I did an ONS no no and didn’t write any posts. That and I had no time because I was working on my SACNAS poster. Regardless, I hold myself accountable and make no excuses (except for the ones I mentioned above).
I’ll post my results later this afternoon from those days. I thought there were issues with the nanodrop, turns out evaporation from my blank cuvette was the culprit. A while ago I measured an appropriate volume to fill my cuvettes so I don’t waste too much yeast. I discovered that 400ul worked well. Thus I filled all my cuvettes to that volume. Additionally the blank cuvette gets for extended periods of time. After about a week or so the volume reduces to around 300ul which I discovered the nanodrop has a difficult time reading at that volume. So therein lies my issues from last week. Today I made a new blank cuvette to take readings from.
On Friday I innoculated some culture from the Thursday (Day 8) samples. Today I measured the results of that growth (72h later) and innoculated new cultures. Here are the measurements:
As I said, I’ll publish the failed results later this afternoon. I have a presentation on Open Notebook Science to give at 1:30 for the PNMGC. Wish me luck!
I setup new cultures for 99% D2O and 50% D2O making this the 4th generation of yeast in D2O. Yesterday I ran out of D2O YPD and had to deal with a smaller batch for the 50% D2O yeast. Today I made some new 99% D2O YPD and inoculated 1ml of each yeast-water type in 9ml of each YPD type. Then I took some measurements.
Today’s Absorption measurements are:
It was interesting to find that the 24h batch of 99% D2O yeast had an absorbance of 2.351 for 2 reasons; 1) that’s a good amount for 24h from what I’ve been observing and 2) because my 1:10 dilution resulted in a much different absorption than 50% D2O yeast (note that they started at a similar value).
When I tried to analyze by eye it was apparent there was a difference in absorbance but it definitely didn’t look 10x less clear. But I did notice that the cells in 50% D2O seemed to be pretty uniformly “dissolved” (I don’t really know what to call that). Meanwhile, the cells in 99% D2O seemed to form larger clumps and the average “particle” size (I’m guessing they are colony clumps) was larger than the clumps in 50% D2O.
I’ll have to analyze this under a microscope and report observations, something that will have to wait until tomorrow or next week.
Another day another dollar… or something.
Ahem…
I set up another culture of D2O and 50% D2O for little yeast to grow in: 9ml of D2O YPD mixed with 1ml of culture from generation 2; 3.5ml of D2O YPD with 3.5ml of DDW YPD and 400ul of 50/50 culture from generation 2. I ran out of D2O hence the reason for the smaller volume than normal. Tomorrow I will try a larger culture.
And here are the measurements from yesterday’s growth and today’s starter measurements:
Yeast Adaptation Day 6. Anthony Salvagno. figshare.
Retrieved 22:05, Oct 02, 2012 (GMT)
http://dx.doi.org/10.6084/m9.figshare.96222
Here are the results. Looks like there isn’t any adaptation yet… I’ll have to figure out how to compare days more thoroughly than eye balling. RESEARCH!!
I’ve broken the DDW sample test tube so these will not be recorded for the rest of the day 🙁
Yesterday’s cultures incubated very well. In just 24 hours the absorbance tripled. I don’t have enough data yet to confirm if that is high, average, or low, but I feel it is pretty decent (which is me taking an optimistic middle ground). Unrecorded yesterday, I started a DDW sample from a single colony for today’s time trial. Let’s check out the stats:
To setup the time trials, I inoculated 1ml of each of the above samples in 9ml of YPD (of each respective type, with 50% D2O being 4.5ml of DDW YPD and 4.5ml of D2O YPD). I will record the growth hourly via the nanodrop.
Yesterday I couldn’t come in to check on the progress of my yeast, so today I’m catching up. Tomorrow I’m scheduling a time trial experiment to check the progress of hourly yeast growth. I’m expanding that experiment to include yeast growth in 50% D2O (mixed with DDW).
But that doesn’t mean nothing grew today. So let’s check out the 48 hour growth (going back to Saturday:
I also started a new second generation D2O sample. There hasn’t been significant growth in the original 2nd gen sample so I pulled a larger amount of culture from the 1st gen sample and put that in a new batch of D2O YPD:
And I followed suit with a second generation of 50% D2O:
The reason for the extra volume of 50% D2O is that I read the pipette wrong and added 5.5ml instead of 4.5ml of YPD broth. Oh well.
To better track the growth, I took so t=0h readings:
So as you can see, my mistake actually put the starting culture absorbance counts at about the same number. That works well and it’ll help better gauge 24h growth.
Until tomorrow…
50/50-72
Sample readings
Tomorrow I’m going to inoculate a larger portion of the original sample into a new 99% D2O YPD sample. And I’ll do the same with the 50/50 sample. I want to go to a larger inoculation volume (right now I”m doing 10ml) of about 25ml, but I need to order more water. That will have to wait until Monday.
Also, I have a suspicion that the initially cold YPD may be hampering the Day 1 growth of all samples, so I’m leaving my YPD sealed, but out of the fridge. And we’ll see what happens with the new samples, that I start tomorrow, come Monday.
The 48h sample reading is 0.997- recall the 24h sample reading of yeast in DI water was 3.05, three times higher absorption!
I replaced that sample in the incubator to measure again tomorrow. I also inoculated a new D2O sample from that one and will record the 24h and 48h growth of cells in that batch. Finally I inoculated some cells (from the 48h D2O sample) and placed them in a 50/50 mix of D2O and DDW. I’ll record those values tomorrow as well.