Monthly Archives: May 2012

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.

KochLab Stuff

Experiments’ Product Page Updated

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I’ve been slacking with keeping up with the equipment that I use in the lab, but I have updated the product page and it should be all up to date with the latest string of experiments. Let me know if you notice I’m missing something.

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)!

D2O1, DDW1, Microorganisms, Yeast

Starter Cultures in D2O, DDW, and DI Water

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Based on the results from yesterday I’ll be carrying out the next phase of the experiment. I set up starter cultures to grow in DDW, DI water, and D2O so I can do hourly time points tomorrow starting early and hopefully getting about 7 hours of data (instead of 5 like yesterday).

I had grown yeast on solid media yesterday and got a bunch of colonies today, so I plucked three colonies and inoculated them in the liquid media stated above (YPD broth).