Final Presentation for REU

Leave a comment

Yesterday afternoon I gave my final presentation for the NSF REU. I’ve posted it on Slideshare for you all to see. It wasn’t real obvious, until I started working on the presentation, how much Anthony and I have done over the last several months. We’ve gotten a lot accomplished!

I have had such a great experience this year that I volunteered to continue coming to the lab this summer. Thankfully Anthony and Dr. Koch aren’t tired of me and agreed to let me stay involved this summer. I hope to be able to work in Koch Lab at least 4 hours a week. I’m interested to see how the e.coli in D2O progresses.

Also, Anthony is really moving and shaking in the Open Notebook Science community. This is really exciting to me.  I’ll be taking my ONS experience with me to my next opportunity…

I’ve been given another NSF internship opportunity, the STEM Talent Expansion Program (STEP), for the summer. [I think that is the right link… the NSF site is down for maintenance until tomorrow night. Let me know if the link isn’t correct]. I’ll be working part-time with Dr. Ramiro Jordan, who seemed open to me keeping an online notebook. Stay tuned for some information about VHDL and circuits.

I am indebted and full of gratitude for all the support and guidance that both Anthony and Dr. Koch gave me this past year. Thank you seems like not enough. Thank you both.

Monitoring e.coli growth

1 Comment

Today Anthony and I are tracking e.coli growth over several hours. His post is detailed, so I wont repeat all the nitty-gritty here (I’ll add a link when he posts his notes).

First we took baseline readings using the Thermo Nanodrop 2000C. Then we made three new batches of diluted e.coli from the batch I ‘Koched’ up yesterday, diluted with LB broth in 1:2, 1:5, and 1:10 parts. Then we put the three batches in the incubator at 37º C and every hour took new spectroscopic readings.

During one of our in-between-spectroscopic-reading-times we ran out to lunch. We went to the Guava Tree Cafe… OMG… absolutely delicious. We both had a stuffed Arepa. I had La del Perro and Anthony had Arepa Pabellón. If you live in Albuquerque you have to try this place. If you don’t live here but get a chance to visit, make sure you eat here at least once. You wont regret it.

Yeast, E.coli day 2

3 Comments

After 12+ hours in the incubators we have growth… well almost. Neither one of the yeast agar plates grew anything. However, both the LB broth and LB agar plate grew e.coli, and the YPD broth grew yeast.

So today we did a few things:

  • We set up two new YPD agar plates using starter culture from the YPD broth that we grew last night. One agar plate is in the 24° C incubator and the other is at room temperature.
  • We decided to grow some more e.coli colonies on an additional LB agar plate using starter culture from the LB broth that we grew last night. It is now in the 37º C incubator.
  • We took spectroscopic readings of our samples using the Thermo Nanodrop 2000C. We used 2mL of the YPD and LB broth and then did the same with 2mL of each starter culture.

Baseline Spectroscopy of Yeast and E.Coli

Yeast Picture

E.Coli Picture

Anther round of yeast and e.coli

2 Comments

Today Anthony and I started preparation for DDW and D2O experiments with yeast and e. coli.  Anthony has a few posts about our past growth experiments. Previously we were successful growing both organisms however, we didn’t get into the DDW or D2O phase of the experiments.

The yeast we are using is S. cerevisiae, g160/2d. We are growing three samples. One sample is being grown in YPD broth that is a mixture of 50mL SIGMA Db/processed water and 2.5g of USB YPD Broth (ultrapure). The other two samples are on 100mm YPD Agar plates from Teknova. One plate is in the incubator with the broth mixture at 24 degrees C. The other plate we are leaving out at room temperature.

We are growing two samples of  DH5α™ e. coli. The first sample on a 100mm LB Agar plate (also from Teknova). The second is in an LB broth mixture of 1g LB broth mixed with 50mL of Dbl processed water, both from SIGMA. Both samples are  in the incubator at 37 degrees C.

We made LB and YPD broth by carefully measuring the indicated amount of broth powder using the lab’s Ohause Adventurer SL balance and small weight boats. We then poured the powder from the weight boat into 50mL beakers and added 50mL of water. We then used magnetic stirrers and a hotplate/stirrer to mix both broths. The LB broth was autoclaved.

Tomorrow we should have some samples ready to go. We are planning on first seeing if we can grow deuterium resistant strains of both the yeast and e.coli. After that we will see how the resistant strains fair in DDW.

Water Bears in the Lab

7 Comments

Wow, it’s been almost two weeks since my last post!  It’s almost spring break, and I’ve been studying like mad for midterms. I’ve been in the lab very little the past two weeks. <sad face>

Monday Anthony and I went on a little excursion to the Bosque looking for tardigrade habitats. Read his post here. It was an interesting experience. Anthony had mentioned tardigrades a few months ago. I did a Google search and found lots of interesting information about these little creatures:

Tardigrade (or water bear), the toughest creature on Earth.

  • They are found everywhere in the world, from the Himalayas to the Artic. They live in Hot Springs and Forests.
  • They can survive in extreme environments because they can pause their metabolism and enter a state of suspended animation, called cryptobiosis.When they are in this state they are in a tun.
  • They can survive without water for over 100 years while in tun.
  • They can withstand 1000 times more radiation than any other living creature known.
  • They can also survive in space (without space suites or spaceships).

All in all they are very impressive little critters.

So why were we looking for them? Well, since I’ve been here, all our research on D2O and DDW has been with tobacco and Arabidopsis seeds. We are now moving into the next phase of research, using yeast and e.coli. One day we were talking about this next phase, and Anthony mentioned that it would be cool to see what effects D2O and DDW have on other life forms. Since Tardigrades are so adaptable, we started looking into getting some for our experiments. Since they are found everywhere we decided to go on a Tardigrade hunt.

The following steps are how to find them (taken from Sarah Bordenstein’s page at Carleton College):

  1. Collect a clump of moss or lichen (dry or wet) and place in a shallow dish, such as a Petri dish.
  2. Soak in water (preferably rainwater or distilled water) for 3-24 hours.
  3. Remove and discard excess water from the dish.
  4. Shake or squeeze the moss/lichen clumps over another transparent dish to collect trapped water.
  5. Starting on a low objective lens, examine the water using a stereo microscope.
  6. Use a micropipette to transfer tardigrades to a slide, which can be observed with a higher power under a compound microscope.

Today we started step 2.

Bosque Samples in water, awaiting for Tardigrade extraction

FTIR – DDW, DI, D2O

Leave a comment

Today I ran FTIR spectroscopy on three ages of DDW, two ages of D2O and the four types of DI that Anthony is using in his current DDW5 experiments.

Many thanks to Stephen Myers for training me on the use of the FTIR, and Dr. Sanjay Krishna for providing me with access to the lab and machine. Stephen was in the lab today and was graciously helpful, as always.

As always I started with a scan of the empty cuvette for the background. Today I kept getting a really strange scan image. Stephen took a look and said confirmed that the scan didn’t look quite right. He changed the Bench Set Gain option to autogain, Anthony cleaned the cuvette, and the next scan came out as we expected it should. After we got a correct background we did a comparison of the previous FTIR background (2/2/2012), today’s background with no autogain correction, and the corrected autogain background. We used today’s autogain corrected background scan for all of our scans today.

background-old-bad-good

Comparison background scans

For each of the following scans we used the same set up as my previous scans from 2/2/2012, using a quartz cuvette and 3µL of the specified water sample.

My first set of scans were three samples of DDW, each opened on a different date (9/6/2011, 1/17/2012, and 2/16/2012). I expected to see a difference because of possible atmospheric absorption of D2O. What I found was they were all pretty much the same the first scan that I ran. Unfortunately, I messed up the save process and had to re-scan the September and January samples. That scan produced a different result for January as shown in the following figure.

DDW second scan

DDW second scan

DDW (second scan) zoomed image

Next I scanned the four types of DI water Anthony is currently using in his current DDW5 experiment (see link). The four types of DI water are:

  1. DI from the Easypure RoDI (Thermohe) machine in our lab (Ro_DI – purple)
  2. CHTM’s DI (CHTM_DI – Red)
  3. Sigma molecular biology grade water (SMol_DI – light blue)
  4. Sigma double purified water (SDP_DI – green)

The results are surprising. The Sigma double purified water’s scan was slightly different than the other three, which were almost identical. This is certainly something to take a look at.

Deionized water scan

Deionized water - zoomed image

Next I scanned two samples of D2O:

  • a bottle opened on 2/16/2012 (red in the scan image)
  • a bottle opened on 11/1/2011 (blue in the scan image)

This scan also produced differing results, possibly from atmospheric absorption (what we had expected to see with the different aged DDW).

D2O scan

D2O scan zoomed

Finally just for comparison, I opened a new window and opened the scan of the February DDW and D2O, the Sigma double processed, and the Sigma molecular biology grade water just to see how the results compared. Interesting that the DDW and Sigma double purified water are identical. Hmmm…

Comparison of DDW, D2O and both of the Sigma DI scans

I’ve uploaded all the raw data onto FigShare with all the images.

My next project is to read up on water frequency and figure out what all the numbers mean. I’ve found a few papers and a website that will probably help shed some light on my very pretty graphs. I’ve ordered two of the papers from the library and the other information is available online.

E.coli – the morning after

16 Comments

Anthony checked the e.coli last night before he went home (after I went to class) and there wasn’t anything going on. He decided to leave the e.coli in the incubator shaker overnight at 150 RPM. This morning when I got into the lab, we checked the test tubes.

The P. Blue is cloudy which would indicate growth, yay!

P Blue e.coli... cloudy indicates that there was growth

The pALS is clear; probably no growth.

pALS e.coli - clear; probably no growth

Thermo Scientific Nanodrop 2000c SpectrophotometerTo accurately measure the number of cells we used a Thermo Scientific NanoDrop 2000c Spectrophotometer.  I’m new to spectroscopy, so for those of you who are also new:

“A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.” ~ Wikipedia – Spectrophotometry

Our method:

  1. Connect the nanodrop spectrophotometer to Anthony’s laptop and start the Nanodrop 2000 application.
  2. L to R: Growth Medium Only, GM & pALS, GM & P BluePrepare the three (3) sample cuvettes, with 1000 mL of LB Broth Growth medium.  The first cuvette has only the growth medium. This will serve as our blank reading (similar to the background reading we take for the Ft-IR).
  3. In the second cuvette we added 1000 mL of pALS to the growth medium.
  4. The third cuvette had an additional 1000 mL of P-Blue.

Now on to the scans:

  1. Click Cell Culture from the main screen of the nanodrop application.
  2. Starting with the “Blank” cuvette (the one with growth medium only), insert the cuvette into the cuvette holder of the nanodrop spectrometer.
    • Check (if not already) Add to Report and Use Cuvette. We used a 10 mm Path Length and no Stir Speed.
    • Click the Blank button (at top of screen).
    • Click the Measure button.
  3. Open the lid, remove the blank cuvette and insert the 2nd cuvette, pALS. Close the lid.
    • Enter a name (upper right of screen) for the new cuvette. We entered pALS.
    • Click the Measure button.
  4. Open the lid, remove the pALS cuvette and insert the 3rd cuvette, P Blue. Close the lid.
    • Enter a name for the new cuvette. We entered P_Blue.
    • Click the Measure button.
  5. Remove the last cuvette and dispose of all the cuvettes properly.

The nanodrop spectrometer software displayed a graph as we measured each sample. The A600 number for each sample was 0.001, -0.01, and 0.714 respectively.

Older Entries

%d bloggers like this: