Finally the starter cultures worked! But not really in the way that I expected. The stuff that was two days old grew and none of the new cultures that I started yesterday grew. Well at least I have something to work with today. Here is my experimental setup:
- Setup of five samples:
- 9ml of DI YPD + 1ml of DI starter culture
- 9ml of DDW YPD + 1ml of DDW starter culture
- 7ml of DDW YPD + 2ml of D2O YPD + 1ml of D2O starter culture (30% D2O sample)
- 4ml of DDW YPD + 5ml of D2O YPD + 1ml of D2O starter culture (60% D2O sample)
- 9ml of D2O YPD + 1ml of D2O starter culture (99% D2O sample)
- 400ul of each starter culture in semi-micro cuvettes – to measuring starting absorbance
- 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)
- repeat B every hour
- Before each hourly measurement you need to blank the nanodrop
- Blank the DI samples with DI YPD (no yeast added)
- Blank the DDW samples with DDW YPD
- Blank the 99% D2O, 30% D2O, and 60% D2O samples with D2O YPD
So of the 6 starter cultures I made (2 of each water type: DI, DDW, D2O) only one sample grew and interestingly and amazingly enough it was the D2O sample! Well today I’m giving it one more go.With the same setup, just using a fresh batch of yeast. The yeast I bought from ATCC.org came in some solution, and I created about 20 aliquots from that solution. So from one aliquot I inoculated 4 new samples (2 DI, 1 DDW, and 1 D2O). And I streaked some from this aliquot one an agar plate.
On top of that I inoculated some of the yeast that had grown in the 1 YPD D2O culture into all the failed cultures. I just want to see if they’ll grow from an established colony.
Let’s see what happens tomorrow.
Yesterday’s starter cultures failed to produce anything, so I am trying again today with my fresh YPD broth. Same protocol:
- Inoculate 1 colony in one of three samples (YPD with DI water, DDW, or D2O).
- Place in incubator/shaker at 30C
Hopefully tomorrow I’ll have better results.
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:
- I still had some YPD from the last time I made broth, so I used that for the starter cultures.
- 10ml of each type of YPD (DI, DDW, and D2O) in test tubes
- Inoculated a single colony from agar media into each sample
- Mix, cover, and incubate at 30C at 150RPM in shaker.
- To make YPD broth:
- Measure appropriate amount of YPD powder for each sample (5g for DI and DDW, 4.6g for D2O)
- Measure out water (100ml of DDW and DI, 92ml of D2O)
- Stir in beaker
- Use syringe and filters to filter large particles and possible microbes in water/broth and syringe into closeable bottles.
- Place in fridge for storage.
- 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!
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!
Hourly Yeast Growth in DDW, DI Water, 30%, 60% and 99% D2O. Anthony Salvagno. Figshare.
Retrieved 21:17, May 30, 2012 (GMT)
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.
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