Tag Archives: setup

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 50: D2O Yeast Time Trials – Setup

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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:

  1. Add 9ml of YPD to 3 test tubes:
    • D2O YPD
    • D2O YPD
    • DDW YPD
  2. 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
  3. Incubate at 30C and shake
  4. 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.

 

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 50

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Wow, 50 days of incubating yeast! Results:

  • D2O yeast gen 39 – 3.065 at 24h
  • D2O yeast gen 40 – 0.739 at 0h
  • DDW yeast – 2.971 at 24h
  • DDW yeast (next gen) – 1.175

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.

 

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 49

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Results:

  • D2O Yeast gen 38 – 3.256 at 24h
  • D2O yeast gen 39 – 0.763 at 0h
  • DDW yeast – 3.249 at 24h
  • DDW yeast (next gen) – 1.026 at 0h

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.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O adaptation day 48

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Results:

  • d2o yeast gen 37 – 3.079 at 24f
  • d2o yeast gen 38 – 0.422 at 0h
  • ddw yeast – 3.039 at 24h
  • ddw yeast – 0.312 at 0h

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.

Arabidopsis Adaptation, D2O Effects on Life, Water Type Effects on Organism Growth

Growing Arabidopsis!

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It was discussed a long time ago that adapting arabidopsis to D2O could reveal some interesting finds. So in an effort to do that I’m going to grow some example plants to test the setup and try and produce seeds.

The one issue I worry about is evaporation. Since this cannot be stopped, evaporation could become a costly component of the experiment (D2O and DDW cost about ~$100 per 100ml). The first experiment will be using DI water. Here is my setup:

  • This is based on the combination of these two protocols:
    • http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/handling.htm
    • http://www.biologyteacher.uconn.edu/tips_arabidopsis.html
  1. I mixed 0.2192g (4.3g/L) of MS salt with 50ml of DI water.
  2. I then put 30ml in a beaker with 0.5g of agar, heated, and stirred. I was supposed to make 1% gel and measured 0.5g for the 50ml that I made instead of for the 30ml that I added to the beaker. When the agar dissolved, I added 10ml of MS salt water to the solution to reduce the gel percentage (now at 1.25%).
  3. I then put the MS agar solution in test tubes: 2 @ 1ml each, 2 @ 2ml each, 2 @3 ml each, and 2 @ 5ml each. I also put ~4ml into some random (but clean) glass jar thing. I bought these a long time ago when I was looking at clear, glass jars for the tobacco and arabidopsis seed DDW experiments. The reason for the various volumes is to determine how much medium the seeds need to grow efficiently and healthily.
  4. While I waited for the MS salt-agar solution to cool (and thus solidify) I poured a bunch of arabidopsis seeds into a petri dish and added some DI water to it. I did not steralize these seeds, because I’m just timing the growth and learning about the pollination process and judging how well the seeds will grow in the lab. For the actual experiments I will steralize the seeds.
  5. Once the growing media cooled, I used a pipetter to collect a few seeds (~5 seeds per sample) and place them in each sample. There are two sets of test tubes (one volume of each set, ie 1ml, 2ml, 3ml, 5ml).
    1. For the first set, seeds were placed on top of the growing medium.
    2. For the second set, seeds were plunged into the gel.
    3. For the random 4ml jar, seeds were plunged into the gel.
  6. Once seeds were set, rubber stops were placed over top of the test tubes to minimize evaporation.

I’m sure this process has tons of kinks, which will be worked out when the real experiment begins, but this is the purpose of an experiment right?

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 47

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So I let the yeast from Friday incubate over the weekend, then there was a power failure. 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.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 44 Time Trial Setup

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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:

  1. 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.
  2. 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.
  3. Incubate at 30C.
  4. 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.

 

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 44

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Results:

  • Yeast in DDW – 1.563 at 24h
  • Yeast in DDW – 3.054 at 48h
  • Yeast in D2O gen 35 – 3.351 at 24h
  • Yeast in D2O gen 36 – 0.742 at 0h

After 48h the yeast in DDW finally equaled the amount of yeast in D2O. I’m inferring nothing, but I’m just sayin’. Anyways I setup the 36th generation of D2O yeast with 9ml of D2O YPD and 1ml of generation 35 culture.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 43

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Results:

  • D2O Yeast gen 34 – 3.097 at 24h
  • DDW Yeast – 1.241 at 24h
  • D2O yeast gen 35 – 0.856 at 0h

Yesterday I did a starter culture and today it’s absorbance is 1.241 in 24 hours. I’m electing to continue incubation of that culture for another 24h and am starting a new culture in 10ml of DDW YPD inoculated via inoculation loop. I also continued the yeast growth of the D2O strain (9ml of D2O YPD and 1ml of culture from generation 34). All three samples are incubating at 30C in my shaker.

D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 42

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Results:

  • D2O yeast (gen 33) – 3.128 at 24h
  • D2O yeast (gen 34) – 0.726 at 0h

I finally got my DDW order so today I did a starter culture of yeast in DDW YPD inoculating a culture from glycerol stock. I also did my usual 9ml of D2O YPD with 1ml of D2O YPD yeast culture (from the previous generation). Tomorrow I hope I can compare the 24h growth of nonadapted yeast in D2O vs adapted D2O yeast. And then the day after that will be a time trial.

Also I made some D2O YPD plates today. So once the starter culture grows I can also compare the growth of colonies on solid media. That should be a fun experiment. Here is my method:

  1. I already had D2O YPD, and I have agarose instead of agar. I added 0.8g of agarose to 40ml of D2O YPD.
  2. I heated the mixture on a hotplate/stirrer to incorporate the agarose. Normally this step is done in an autoclave, but I can’t use that because I don’t want to add more H2O than necessary to the D2O (d-exchange).
  3. Pour 15-20ml per plate. I made enough for 2 plates.