Tag Archives: yeast

D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Time Trial Growth: Starter Culture Setup

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This is, I believe, the fourth trial of doing yeast growth time points. I setup the starter cultures and made some new YPD broth and here is how I did it:

  1. I still had some YPD from the last time I made broth, so I used that for the starter cultures.
    1. 10ml of each type of YPD (DI, DDW, and D2O) in test tubes
    2. Inoculated a single colony from agar media into each sample
    3. Mix, cover, and incubate at 30C at 150RPM in shaker.
  2. To make YPD broth:
    1. Measure appropriate amount of YPD powder for each sample (5g for DI and DDW, 4.6g for D2O)
    2. Measure out water (100ml of DDW and DI, 92ml of D2O)
    3. Stir in beaker
    4. Use syringe and filters to filter large particles and possible microbes in water/broth and syringe into closeable bottles.
    5. Place in fridge for storage.

Notes:

  • There was a bunch of mold in some samples of the yeast colonies, so I threw those out in biowaste. In some other samples, the colonies were overgrown so I also put those in biowaste. I started another agar media colony (just streak a colony onto a ypd agar plate, pre-purchased) to replenish my stash.
  • I used only 92ml of D2O because that is all there was in the bottle. Since I’m using 50g/L ypd powder that means I need 4.6g of powder for my liquid media. Hence the numbers provided above.

Tomorrow begins the time trials, yay!

E. Coli, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast and E. coli Growth: What’s next?

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There are several things I can do with the yeast experiments:

  • Another but longer time trial experiment (data every hour)
  • Time Trials in larger quantities: maybe 100ml or 50ml – I want to see if the growth in such small volumes affects the measurements any
  • Adapt yeast to D2O and then grow in DDW – I’ll have to do cultures daily in D2O and see if they begin to grow faster in D2O over time. I’m thinking this will let me know they’ve adapted to their environment. I can also try to grow cultures in less D2O (like 10%) and incrementally increase it over time to see if that causes adaptation. Both methods seem viable.

I’m open to suggestions as to what to do next as well.

As for E. coli, those experiments are very inconclusive even in 99% D2O and I wouldn’t have the foggiest as to how to deal with this. Suggestions would be much appreciated here. Ayuda me!

D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Hourly Yeast Growth Trial 3: Results

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Via Figshare:

Hourly Yeast Growth in DDW, DI Water, 30%, 60% and 99% D2O. Anthony Salvagno. Figshare.
Retrieved 21:17, May 30, 2012 (GMT)
hdl.handle.net/10779/03ec08019a22d82c66167d5e7d914de5

This experiment went much better and more predictably. Yeast (unlike E. coli) is more sensitive to deuterium content and grows accordingly. Interestingly (in this experiment) the DDW sample exhibited more growth. I’ll have to experiment on this a bit more.

It should be noted that while it appears that pure D2O grows much slower than the rest (cause it does), I also started with a much lower amount of cells. The cells did not grow well in the starter culture, and they didn’t grow well here either. See the data (or the live results) for starting absorbance readings.

D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast in DI, DDW, 30%, 60%, and 99% D2O Trial 3: Setup

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Yesterday I made starter cultures in D2O, DI water, and DDW. Today I’m doing the time trial experiment and here is the setup:

  1. Setup of five samples:
    1. 9ml of DI YPD + 1ml of DI starter culture
    2. 9ml of DDW YPD + 1ml of DDW starter culture
    3. 6ml of DDW YPD + 3ml of D2O YPD + 1ml of DDW starter culture (30% D2O sample)
    4. 3ml of DDW YPD + 6ml of D2O YPD + 1ml of DDW starter culture (60% D2O sample)
    5. 9ml of D2O YPD + 1ml of D2O starter culture
  2. Measuring:
    1. 400ul of each starter culture in semi-micro cuvettes – to measuring starting absorbance
    2. 400ul of each timed culture (the samples to be measured over time) in semi-micro cuvettes – to measure the time=0 value (should be close to 1/10 of the starting measurements)
    3. repeat B every hour
  3. Blanking:
    1. Before each hourly measurement you need to blank the nanodrop
    2. Blank the DI samples with DI YPD (no yeast added)
    3. Blank the DDW, 30% D2O, and 60% D2O samples with DDW YPD
    4. Blank the D2O samples with D2O YPD

 

D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Starter Cultures: D2O, DDW, and DI YPD

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I just started three new starter cultures for another timed experiment tomorrow. Hopefully this time it goes better than last time.

Setup:

  • 10ml of YPD broth in test tubes
  • The water component of each test tube is different, one is DDW, one is DI water, and the other is 99% D20.
  • The YPD was made last week and stored in the fridge.
D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast hourly growth in DI, DDW, and 30%, 60%, 90%, and 99% D2O

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Results:

Yeast growth in DI, DDW, and 30%, 60%, 90%, and 99% D2O. Anthony Salvagno. Figshare.
Retrieved 21:20, May 24, 2012 (GMT)
hdl.handle.net/10779/caa57855586213d79eea5576f8da23d0

Notes:

  • I don’t think this is a reliable data set. Several of the samples actually read a lower absorbance after the first hour than they initially do. And then they all dip again at hour 4.
  • I noticed a considerable amount of cell settling after hour 3 on the bottom of each test tube. I mixed prior to reading the absorbance values, but this is likely to skew the results. Next trial I will have to mix before reading every hour.
  • The data between DI, 30%, 60%, and 99% D2O look consistent with Tuesday’s results, but it scares me that the DDW and 90% D2O are completely out of whack.
D2O1, DDW1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast hourly growth in DI, DDW, and 30%, 60%, 90%, and 99% D2O: Setup

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Yesterday I set up starter cultures for this experiment using D2O, DDW, and DI water as the basis for the liquid media. Today I’m running the time trial experiment again, but this time with a bit more of a spectrum in water amounts. Here is the setup:

  • I am using 6 samples: DI water, DDW, 30% D2O mixed with DDW, 60% D2O mixed with DDW, 90% D2O mixed with DDW, and 99%D2O.
  • For the mixes:
    1. add D2O, add DDW, add 1ml starter culture from DDW (1:10 dilution).
    2. Example: 30% D2O in DDW is 3ml D2O, 6ml DDW, and 1ml DDW starter culture
  • For the other samples:
    1. DI: 9ml of DI YPD, 1ml of DI starter culture
    2. DDW: 9ml of DDW YPD, 1ml of DDW starter culture
    3. 99% D2O: 9ml of D2O YPD, 1ml of D2O starter culture

It should be noted that the 99% D2O starter culture was almost completely translucent. By this I mean it looked like there was no growth in the sample at all. Results that I’ll post later (when the experiment is complete) will show that this is indeed true, with an absorbance of 0.521 (compared to ~2.1 in both DI and DDW starter cultures, 4x the cell growth!).

I’ll be taking growth readings every hour via the nanodrop in micro cuvettes (til about hour 4) and then switching over to semi-micro cuvettes (because I’ll run out of micro cuvettes).

Love,

Ant

KochLab Stuff

Using cuvettes in the nanodrop

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I did a mini-study this morning to find out what the minimum volume needed is to get an accurate reading in the nanodrop. I have 2 different cuvettes (semi-micro and micro) and I wanted to impact the cultures the least so I would like to use the micro cuvettes.

image
From left: 400ul in semi-micro cuvette, 200ul in micro cuvette, 500ul in semi-micro cuvette

I used 5 semi-micro cuvettes and 2 micro cuvettes. I put increments of 100ul starting at 100ul in each cuvette (100 -> 500ul in the semi-micro and 100 and 200ul in the micro cuvette). At and above 400ul the nanodrop was able to effectively and consistently read the absorbance of the semi-micro cuvette (verified because the readings for 400 and 500ul were identical). For the micro cuvette, I looked at the profile (image above) and saw by eye that the height of the meniscus of the liquid media was roughly equivalent to the height in the semi-micro cuvette with 500ul. So I put this in the nanodrop and got an equivalent absorbance reading.

So in summary:

  • For semi-micro cuvettes (and the Thermo Nanodrop 2000c), volumes of at least 400ul or more are sufficient for consistent readings.
  • For micro cuvettes, volumes of 200ul are sufficient for consistent readings.

The End.

D2O1, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Growth at RT: Data and comments

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Via figshare:

Yeast Growth at RT in D2O and DI water. Anthony Salvagno. Figshare.
Retrieved 18:10, May 23, 2012 (GMT)
hdl.handle.net/10779/736ebb6f977250f55e9d2bbb12f4f796

First let me explain the experimental setup:

  • This is a carry over from yesterday’s experiment. With the setup here.
  • Every hour I took data from the cultures I inoculated. I put 500ul in semi-micro cuvettes to measure in the nanodrop.
  • After I take data, typically I store the measured cuvettes at RT on the lab top in a cuvette holder (the cuvettes are sealed with PE caps).
    • Originally I did this because I wanted to take a picture to compare the growth after the full experiment was completed, but I noticed with e. coli that the cultures continue to grow at RT, so a picture wouldn’t reveal anything or be accurate.
  • After this experiment I decided to use the nanodrop to compare the growth at RT from all the samples (with the more recent samples having less time to incubate at RT for obvious reasons).

Linked above is the results of the experiment with the excel file I used to compile the data.

Now it’s time for some notes:

  • Originally I had compared the growth of these samples to the original data. But realized that the comparisons weren’t valid because each sample grew over 5 hours and the samples were independent of each other at this point.
  • It is interesting to note that the D2O sample is almost the same in every sample, except in hours 4 and 5 where the growth is more from incubation at 30C than RT (~25C). But even those two data points are almost identical.
  • The DI sample has more steady growth in each sample. I should compare these values to what they were when they were removed from the incubator.

I don’t know if this data is useful but I thought it was interesting so I thought I’d share it with the world. If you have any suggestions for this particular experiment let me know (comment, twitter, email, snail mail, etc)!