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!
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.
Feel free to check in every hour (refresh this page)!
Yesterday I made starter cultures in D2O, DI water, and DDW. Today I’m doing the time trial experiment and here is the setup:
- Setup of five samples:
- 9ml of DI YPD + 1ml of DI starter culture
- 9ml of DDW YPD + 1ml of DDW starter culture
- 6ml of DDW YPD + 3ml of D2O YPD + 1ml of DDW starter culture (30% D2O sample)
- 3ml of DDW YPD + 6ml of D2O YPD + 1ml of DDW starter culture (60% D2O sample)
- 9ml of D2O YPD + 1ml of D2O starter culture
- 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, 30% D2O, and 60% D2O samples with DDW YPD
- Blank the D2O samples with D2O YPD
I just started three new starter cultures for another timed experiment tomorrow. Hopefully this time it goes better than last time.
- 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.
Yeast growth in DI, DDW, and 30%, 60%, 90%, and 99% D2O. Anthony Salvagno. Figshare.
Retrieved 21:20, May 24, 2012 (GMT)
- 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.
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:
For the other samples:
- add D2O, add DDW, add 1ml starter culture from DDW (1:10 dilution).
- Example: 30% D2O in DDW is 3ml D2O, 6ml DDW, and 1ml DDW starter culture
- DI: 9ml of DI YPD, 1ml of DI starter culture
- DDW: 9ml of DDW YPD, 1ml of DDW starter culture
- 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).