Microbial Constellations

On Monday, we viewed the aforementioned epithelial cells under a fluorescence microscope.  These cells were grown to express green fluorescence protein (GFP), which can be viewed under the microscope, as seen below.  Pictures need to be taken quickly, lest the cells begin dying and deform/burst.  To achieve a desirable picture, you must navigate about the slide using knobs attached to the stage below the screen until you reach a patch that displays a good amount of GFP expression.  Take a picture (there is a feature for that on the screen).  You must then turn on the regular trasnmittance so that a picture of the cells as they are (not lit up) can be taken.  This is layered over the previous GFP picture and settings are adjusted to get as clear a picture as possible.  Looking at these clusters of cells, seeing them twinkle, reminded me of what I see when I look up at night (in Cambria, where little air and light pollution exists).

There is great contrast between the expressing cells and the background.  If you were to see the image up close, you'd see that the nucleus of the cell is lit up, with dull cytoplasm surrounding it and the dark outline of the membrane surrounding that.  The nuclei on these are large, since these are mammalian cells, so the bright green takes up most of the cell.

There is quite a bit of background noise in this one.  The contrast should perhaps be adjusted.

This is as good as my mentor could this picture to be.  There is a clear difference between the background and the lit up cells.  If you look closely, you can see microtubules stemming from the cells.  There are some going through mitosis as well.  Pretty cool.

Today, we pressed drug pellets.  I weighed so-and-so milligrams of powdered drug onto a folded wax paper sheet and handed it over to the college intern working with me to press it into a pellet.  it was repetitive and sometimes frustrating work, getting the weight to be within .2. milligrams of that desired. It gave me time to think about time and black holes... thank you Mrs. Pittman and Brian Cox.

After that, I worked on the microtome and talked to Paul about a biofilms project he is working on. Quite fascinating.  

 The glass knives are at top, the resin blocks below them to the fight, a bunch of parts below those, and a package of razors below those (the one with the yellow label.

January Update

I went to mentorship twice this week: Monday and Friday.

Monday was a 9:30am to 3:00pm day beginning in the tissue culture lab, where mentor was handling a flasks of epithelial cell culture under the ventilation hood.  A little bit about tissue culture: you cannot keep your cells in the same media for too long lest they die.  As the cells grow, it can get a bit crowded, as you can imagine, and the cells must be split up.  Furthermore, the cells tend to start sticking to the walls of their flask after a while.  To detach them, they are treated with trypsin, which hydrolyzes the peptide bonds in the protein attaching the cells to the flask wall.  The trypsin must be deactivated with a neutralizing medium, as prolonged exposure to it will damage the cells.  The cells are then spun down, resuspended, and split.  In this case, my mentor split the cell into three flasks, giving them fresh medium to feed on.

cells in growth medium

After this culture business, we attended a meeting on general lab safety, then began on tar extraction.  It was glorious, magical, fantastic - like the last I participated in it.  We pounded tar to a powder!  Upon completing most of the process (we ended at a lengthier step and never proceeded to the next), I cleaned up at a lab adjacent to ours, had a chat with Mr. Webster, and worked with an intern as she pressed pellets of a drug we are looking into.  It's another sustained-drug-release project.  She, in turn, showed me how to operate the solicitor.  It's quite simple.  You must place whatever you are sonicating into a falcon tube, fill the tube with water, simply place it in the solicitor (pretty much a container of water) and turn the thing on.  Low frequency sound is generated.  This is one form of maceration - the breaking up of something.  Maceration can be chemical (ex: acid) or mechanically (ex: metal pestle, sonication).  

My day ended, then.  

On Friday, I worked the microtome.  I took some pictures with my cell-phone, which I shall add here shortly (upon figuring out how to transfer photos from my phone to this computer…).  I mounted a resin block onto the segment arc, which I attached to the trimming block, and that onto the stage trimmed the block with a metal blade first, then a moved on the glass, fixing the segment arch onto the cantilever arm.  The actually cutting of the resin was a challenge for me, though, and it came out all messy and rough.

End of the day surprise: free books!  There were just piled there at the end of the hallway by the microtome.  I 'd heard that best ones were taken days ago… but I managed to find Shakespeare!  Hopefully there are still some left for me to rummage through next week…    

As far as independent learning goes, I found a courses on astrobiology and climate change on Coursera.  The latter is a possible independent component idea.  We shall see.

Third Interview Questions

Here are my 10+ questions.  

1. What is the most useful application for extremophile research in addressing global climate change?

2. What are the most pressing problems in the addressing of global climate change today (roadblocks, etc.)?

3. How can these problems be solved?

4. What is it about extremophiles that makes them particularly useful for the aforementioned purpose?

5. What possibilities are there in bioremediation?  How about in hydrocarbon pollution clean-up in particular?

6. Which extremophiles in particular that have proved to have potential in such advancements?

7. How could extremophiles be used in biofuel production?

8. Which extremophiles in particular that have proved to have potential in this field?

9. What do we need to understand more in order to tackle global climate change?

10. How could extremophile research contribute to the development of this understanding, perhaps of a clearer global climate model?

11. What extremophiles show particular interest in this development?

12. How far has extremophile research come? Is there is still a long way to go before any of it can be applied?

13. What papers or journals you would recommend I use to stay up to date on developments involving the application of extremophile research to addressing global climate change?

14. What people or organizations in particular are involved in work related to my senior project?

Blog 11: Mentorship 10 Hours Check

1.  I am mentoring at the Oak Crest Institute of Science in Pasadena.

2. Manjula Gunawardana, my mentor

3. I have completed 30 hours of mentorship.

4. Mentorship always holds surprises for me.  I began with culturing shrimp with an undergrad who interned over the summer.  From there, I received the opportunity to extract the RNA from my very own cheek cells, converting it to cDNA (otherwise known as ssDNA (single-stranded)) afterwards.  Next came OD readings and DNA extraction from yeast, preparing samples for electron microscopy and operating an ultramicrotome, autoclaving trash, coating pods, and, finally, witnessing a fellow aspiring scientist pipette for the first time.  There was also SCCUR and the thrill of presenting an exciting shrimp bioassay on potentially pharmaceutical plant extracts.  We'll see what 2014 at Oak Crest holds.

5. I will do that soon.

Senior Project: The Holiday

1. I read two of the papers that my mentor gave me and went to mentorship on January 3.  I also updated my independent log and began looking into some professors at Cal Poly Pomona and Pomona College who could perhaps be future interviewees of mine.  

2. I know, I know - that's only 10% of my "20 friends", but hey, I was busy tide pooling at Cambria.  In fact, it reminded me of the complex ecologies that exist in our world.  In one little pool, there were hermit crabs fighting over a shell, anemones chilling, a sea snail struggling to reach water with another sea snail attached to its back, a tide pool sculpin flitting about - just the general give and take of nature.
The same exchanges can be seen at the microscopic level.

tide pool sculpin

Also present on those rocks were smudges of oil, perhaps from leaks from the S.S. Jacob Luckenbach - a cargo ship loaded with 457, 000 gallons of bunker fuel.  Cambria marks the southern tip of the Monterey Bay National Marine Sanctuary - governed by tight regulations and home to a marked perfection I've had the privilege of being witness to many times.  THIS is what all coasts should be like.  They don't deserve oil spills.  THIS is why I chose an environmental focus for my senior project.

I couldn't find any photos of my own, so here is the next best thing.  Behold: moonstone beach.

Anywho...
At mentorship, I worked with a community college who was pipetting for her first time.  I showed her how to tap the 96-well plate to pull droplets off the sides of the wells, and operate the UV Vis.  My mentor helped out as well, showing us his wicked pipetting skills and giving tips on how to deal with bubbles (the bubbles were from aspirating the liquid - that means pumping it up and down to mix it (in this case, 'twas a mixture of BSA and sodium azide)).    

Such fashionable pipette tips.  Behold their glory!

Reading "Microbial Diversity in Natural Asphalts of the Rancho La Brea Tar Pits" and "Advantages and Limitations of Quantitative PCR (Q-PCR)-based Approaches in Microbial Ecology" exposed me more to terminology I will become familiar enough with over the course of my project.  It's all about gaining intuition and becoming comfortable with my topic.
There are a few Cal Poly professors who may be of some interest - sort of.  At Pomona College, there's this one professor who has a special interest in the microbial ecology of mud volcanoes.  He might be a future resource... 

3. I would talk to my mentor, and perhaps the aforementioned E.J. Crane of Pomona College.  There's also CalTech (only a few minutes from my house, actually), home to a geobiology department and Victoria J. Orphan (whom my mentor mentioned over the summer).  She's a molecular microbial ecologist, primarily, and another potential interviewee.