Tag Archives: setup

D2O Adaptation 3, Microorganisms, Water Type Effects on Organism Growth, Yeast

Adding Ampicillin to YPD

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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.

D2O Adaptation 3, Microorganisms, Water Type Effects on Organism Growth, Yeast

20% D2O YPD Agar Plate

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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 3, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast in 20% D2O Time Trial

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Setup. Today 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:

D2O Adaptation 3, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Try 3 – Project Plan

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Now that the lab is clean, I’m going to collect my thoughts and write a plan to follow. While the first two experiments were unsuccessful, there were some interesting results that can’t be disregarded. But in the pursuit of science more results are required! So let’s get this party started…

D2O adaptation: Based on the paper I read yesterday, it seems that a slow adaptation is the right way to go. Yeast grown in pure D2O seemed to endure too much stress to adapt and the adaptation might be way slower compared to a progressive adaptation plan. So each week I’ll increase the amount of D2O by 20% starting with 20% D2O YPD:

  1. 20% D2O
  2. 40% D2O
  3. 60% D2O
  4. 80% D2O
  5. 100% D2O

Plan:

  1. Every Monday I plan on running a time trial experiment comparing the growth of yeast in DI YPD to the increased D2O YPD concentration and to the previous D2O concentration.
  2. Every Friday I’ll run a time trial of the yeast growth in that week’s D2O concentration.
  3. Every day, yeast will be provided new medium for growth and daily nanodrop readings will be recorded to analyze the 24 hour growth. Also a microscope analysis will also be done to ensure there is no contamination.
  4. Weekly glycerol stocks will be created in case of contamination.
  5. Agar plates of each D2O concentration will be created to analyze colony morphology. Inoculation onto solid media will take place each week, and right now I’m not sure when the best day to do this is, but I’m thinking Wednesdays might be best.

 

D2O adaptation, D2O Adaptation 2, D2O Adaptation 3, Microorganisms, Water Type Effects on Organism Growth, Yeast

Daily Yeast Growth Protocol

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These are my protocols for the D2O adaptation experiments. I’m making this post so I don’t need to write a post daily unless something out of the ordinary happens.

Making YPD:

  1. Use autoclaved beakers and bottles for water handling and storage.
  2. Measure water volume: D2O usually is supplied as a little over 90ml per bottle, DDW is about 100ml per bottle, and DI is readily available in whatever quantity needed.
  3. Measure YPD powder and add 5g per 100ml of water.
  4. Add a clean stir bar and stir (using hotplate/stirrer) until YPD is fully dissolved. For D2O and DDW stir at a low speed to minimize air mixture with solution.
  5. Using a syringe and a filter, filter the YPD into the bottles. This step is necessary for D2O and DDW YPD to ensure minimization with H2O/D2O contamination. For DI YPD autoclaving is sufficient.
  6. Label bottles and date.

Daily Prep:

  1. For starter cultures, add 10ml of YPD to an autoclaved test tube. For daily measurements, add 9ml of YPD.
  2. Inoculate yeast in liquid media. For daily measurements, inoculate 1ml of culture from the previous generation’s water type (ie add 1ml of 99% D2O yeast to 99% D2O YPD).
  3. Add to incubator/shaker and incubate at 30C and 185RPM.

Nanodrop Measurement:

  1. Aliquot a minimum of 400ul of sample into semi-micro cuvettes. Normally I measure at 0 hours and at 24 hours (or every 24h thereafter for prolonged experiments) for daily measurements and hourly starting with the 0h measurement for time trial experiments.
  2. Blank the nanodrop with pure YPD and make sure to press the cuvette checkbox (I always forget this!).
  3. Measure each sample.
Arabidopsis Adaptation, D2O Effects on Life, Water Type Effects on Organism Growth

Arabidopsis growth try 2 setup

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20121203-151328.jpg

Since I had leftover MS salt-water mixture leftover from the first experimental setup, I decided to use the remaining bit to make this batch. I did add some water since last time I had over 1% gel. I added about 5ml to the remaining solution, which is some unknown amount. Obviously this isn’t the best experimental setup, but I essentially just want to see if I can grow plants so accuracy isn’t too important.

I have received some replies from some UNM Biologists and hopefully can meet with some students this week to work out some experimental details. More information to come as I know it.

D2O Adaptation 2, Microorganisms, Water Type Effects on Organism Growth, Yeast

Yeast Adaptation Day 3

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My original yeast is still going in D2O. There hasn’t been much growth in the past 48 hours. And to hopefully achieve a faster/more efficient form of adaptation, I’m growing yeast in slightly increasing amounts of D2O. Today I started growing in 20% D2O (mixed with DI water to save money).

After 48 hours of growth the yeast in DDW measured 3.327 in the nanodrop.

Also I noticed that the YPD in DDW aggregated. This took about 12 days. I’ve never had this happen in D2O, except one time it did in a 1ml amount in a cuvette. That took about 14 days. And it was next to a heat source during that entire time. So it would be interesting to test the aggregation affects of YPD in DI/DDW and D2O. If I can achieve an adapted form of yeast, growing in D2O YPD could be beneficial all around (cost effective).

D2O Adaptation 2, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Try 2: Day 1

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Because I’m 90% confident that the adapted yeast was actually e. coli (based on images and smell) I’m going to restart the experiment. I started by cleaning the incubator/shaker. Might as well try and minimize e. coli outbreak…

Setup:

  • Add 9ml of D2O YPD (all that I had left) to test tube.
  • Inoculate a colony from yeast grown on D2O solid media.
  • Add 9ml of DDW YPD to test tube
  • Inoculate colony from yeast grown on D2O solid media.
  • Place in incubator at 30C and 185rpm

I also made a fresh batch of D2O YPD:

  • 4.6g of powder YPD
  • 92ml of D2O
  • stir until dissolved
  • filter (2um) into bottle
D2O adaptation, Microorganisms, Water Type Effects on Organism Growth, Yeast

D2O Adaptation Day 51

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No results today 🙁 I accidentally disposed of the samples from yesterday after inoculating the samples for today. And there is no data today because the inoculation used an inoculating loop, so the absorbance wouldn’t be noticeable. But the setup is 10ml of YPD (3 samples, two DDW YPD and one D2O YPD) with an inoculation (using the loop) from the previous generation. The reason for the 3 samples is because I want to see if after a few generations, the D2O adapted yeast revert back to the wt yeast, so in addition to a sample of DDW yeast and D2O yeast, I’m doing the D2O adapted yeast in DDW. We’ll see what happens.