Tag Archives: setup

80% D2O YPD Solid Media

  1. Water (already prepared and antibiotic added):
    • 32ml D2O YPD
    • 8ml DI YPD
  2. 0.8g agar
  3. Stir and heat on hotplate
  4. Pour 15-20ml into petri dish and allow to cool
  5. seal with parafilm

60% D2O YPD plates Setup

  1. Water (already prepared and antibiotic added):
    • 24ml D2O YPD
    • 16ml DI YPD
  2. 0.8g agar
  3. Stir and heat on hotplate
  4. Pour 15-20ml into petri dish and allow to cool
  5. seal with parafilm

New YPD made

I just ran out of my last batch of both DI and D2O YPD. This time I am making the protocol with 500ul of Ampicillin added (460ul added to D2O).

Yesterday I added amp to my 10ml test tube sample and it seems to have killed the bacterial infestation without impacting the yeast growth. Normally when there is bacterial growth, the yeast settles and the bacteria floats in solution. This morning the solution was clear and the yeast was nicely nestled at the bottom.

Adding Ampicillin to YPD

So I’ve noticed that no matter what, there is some bacterial contamination, and based on an experiment I did with the reconstituted yeast, it seems the contamination exists there. After doing some research, it seems I can add antibiotics to the YPD without affecting yeast growth and preventing bacterial growth. So today I’m doing just that. I’ll take a picture of tomorrow’s culture to reveal whether or not this method works against the bacteria, and if it impacts the yeast growth.

It turns out that I have ampicillin as a powder. So I added 20ml of water to it to bring it to a concentration of 10mg/ml. According to a protocol I found on my old stomping grounds (openwetware.org), I need to add 50ul to 10ml of YPD to get a final concentration of 50ug/ml.

20% D2O YPD Agar Plate

I made 2 20% D2O Agar Plates:

  • 32ml of DI YPD
  • 8ml of D2O YPD
  • 0.8g agar
  • stirred and heated until agar dissolved
  • 15-20ml of agar solution into petri dishes

I allowed the plates to cool and sealed one and put it in the fridge for later use. The other went streaking and is in the incubator at 30C.

 

D2O Adaptation Live Results

Setup

Here are my daily results. Each week I will be increasing the % of D2O in solution, starting at 20% D2O.

Yeast in 20% D2O Time Trial

Setup.┬áToday I”m doing the time trial and I setup 3 samples, following my protocol:

  1. Yeast in DI YPD
  2. Yeast in 20% D2O YPD
  3. Yeast in 99% D2O YPD

Live Results: