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

Day 36 Time Trials: Setup

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Yesterday I created starter cultures for today’s time trial experiment. Today’s goal is to analyze the growth of the (potentially) D2O adapted yeast and compare it to the growth of non-adapted yeast grown in D2O, 50% D2O, and DDW.

I started by measuring the growth (absorbance) overnight from 6 samples of yeast grown in:

  1. DDW (from adaptation experiments) – 3.075
  2. 50% D2O (adapted from adaptation experiments) – 3.079
  3. 99% D2O (adapted from adaptation experiments) – 2.950
  4. DDW (from glycerol stock) – 1.177
  5. 50% D2O (non-adapted from glycerol stock) – 0.556
  6. 99% D2O (non-adapted from glycerol stock) – 0.041

I created the time trial cultures as follows:

  1. 9ml of each water type in test tubes for a total of 6 samples (2 of each water type)
    • DDW
    • 50% D2O (4.5ml DDW, 4.5ml 99% D2O)
    • 99% D2O
      • because the non-adapted yeast in 99% D2O grew in such a small quantity I am just using the entire culture as a starter culture
  2. For the non-adapted samples I added 1ml of each culture to each water type, except for the 99% D2O sample which I explained above (3 samples total)
  3. For the adapted samples I added 0.4ml of each culture to each water type (3 samples total)
  4. Measure the samples hourly via the nanodrop in semi-micro cuvettes filled to 400ul.

The reason for the different amounts of culture in each sample is to get the 0h absorbance reading as similar as possible. This way it will be easier to compare growth rates. Here are the starting absorbance values:

  1. DDW (from adaptation experiments) – 0.259
  2. 50% D2O (adapted from adaptation experiments) – 0.222
  3. 99% D2O (adapted from adaptation experiments) – 0.228
  4. DDW (from glycerol stock) – 0.160
  5. 50% D2O (non-adapted from glycerol stock) – 0.059
  6. 99% D2O (non-adapted from glycerol stock) – 0.031 (remeasured)

It is interesting to note that the non-adapted yeast grown in 99% D2O struggled to grow as a starter culture, like I’ve witnessed countless times (ok maybe like 10 times). But the adapted yeast grew just as well as the DDW yeast under the same conditions.Further proof of adaptation? I think it helps the case.

Recall the difference between the sets of samples is that 3 samples were inoculated from already existing cultures and the other 3 were inoculated from glycerol stocks. Both were inoculated the same way otherwise (ie with an inoculating loop).