CRUNCH TIME!

Well, here we are end of the semester and I'm still holding strong. As one of my professors said today, "It's all over except for the crying." That made me choke on my coffee!

This week was focused more on wrapping things up for the end of the semester, so there was no progress made on my continuing research. There were a few conversations about possible modifications to my DNA extraction protocol, but this will hold for next semester (if i am lucky enough to continue in this program). My samples are all stored at -80 degrees celsius, they will hold just fine.

The bulk of this week was spent doing the "spit and polish" routine on papers due in my classes and of course the research paper on my bacterial unknown. That one kind of grew exponentially, it is much longer than I anticipated it would be. I also have developed my first ever powerpoint series for my oral presentation of research results on Friday. I was evidently worried about nothing with it. Powerpoint slides are much easier to develop than I anticipated, and that was a big relief! No I can just focus my stress on giving the actual presentation. I have to share a suggestion i was given about speaking in front of groups. I have a pretty high prescription for corrective lenses, and the suggestion was made that I not use my contacts and wear my glasses the day of the presentation, and that I should take them off just before presenting. The theory is, if I cannot see the audience, then I cannot be nervous about them. hmmm...

A little humor to wrap up on, Let us hope that this is not the case...

I would also like to give a special thanks two these to for helping me make it through another semester!

Enjoy the Holidays, and see you next time...

Well then...

Well, there may be some issues with my extraction technique. As the protocol I used will be undergoing some drastic changes, and could possibly be scrapped completely for another method, I will give just an overview of what is going on.

The above sample of leaf material that I collected was ground to a slurry in a lysis buffer solution that served to extract the DNA from other plant cell material. DNA is a negatively charged molecule and there are positively charged "beads" in the solution that adhere to them. The entire solution was then centrifuged to separate the beads and DNA from the heavier plant material. After filtration and a wash with isopropanol, I should have had a viable sample of DNA to run gel electrophoresis.

In gel electrophoresis, DNA is loaded into an agarose gel and current is applied (in this case 120 volts for 30 minutes). As I mentioned before DNA holds a negative charge and by placing it in the gel near the negative probe, it is drawn through the gel towards the positive probe. As DNA travels through the gel segments with more base pairs (Adenine with Thymine, and Guanine with Cytosine) will travel much slower than those with fewer base pairs, and the bands can be then observed and recorded.

This is the gel box I used. Apologies for the poor quality picture, I believe I deleted the wrong picture.

In my case, this did not go so well. The DNA sample did travel, but it went the wrong way! THe sample traveled to the negative end of the gel. This indicates to me that the positively charged beads used in the extraction actually made it through the filtration and wash process and were still bound to the DNA and pulled it to the negative pole. After discussion with Anil, and Matt, we have a couple different ways to change our extraction protocol. We think that the protocol we used was designed more for animal cells than plants, and the filtration wasn't up to par with the plant material.

I am not sure when I will continue with the extractions as the weight of the end of the semester is upon us. My research paper on the bacterial identification draft needs some polishing into a final draft, and I still need to develop an oral presentation of it as well (not to mention papers due in my other classes and impending finals). This extended Thanksgiving weekend will not be quite as relaxing as those passed...

HAPPY THANKSGIVING ALL!

In my last post, I left a question hanging: where is the line between the separate clone entities and one connected organism. Well, after some digging I have found another species that clones in much the same fashion: Populus tremuloides, the Quaking Aspen. Not only has it exhibited the same cloning adaptation, an example of it also happens to be THE WORLD'S LARGEST LIVING ORGANISM! According to the US Forest Service, Pando (as the grove is known), covers roughly 106 acres, weighs in at an estimated 13 million pounds and consists of over 40,000 individual trees that are, you guessed it, CLONES! These clones sprout form the root system in much the same manner as my little old Palo Verde.

"Pando" courtesy of USFS

This will definitely help me in laying out the proof of cloning (along with good DNA testing results, of course). I am currently trying to track down a couple of peer reviewed journal entries that relate experimental data with the aspen in much the same fashion as I am beginning to do with the Palo Verde. Unfortunately I have only been able to read through the abstracts as of yet and i want the data. A little time and effort and I should be able to find similar research. 

As far as my experimentation goes, today I conducted my first DNA extraction from one of the many samples I have collected. Next week I should have some gel results that will show wether or not I was successful at extraction. I will leave explaining the method of extraction for next week's post where I will fully explain the entire process from extraction to running the gels.

I think I'm a clone now...

Well, the ball is finally rolling on my research project. I will be looking at plant species that are known to produce sexually and attempting to show the fact that they do reproduce through cloning as well. The first species I will be looking at is Palo Verde.

When attempting to decide what topic I would cover for my project, i came to a decision that I would like to learn more of the nuts and bolts of genetic analysis. After extensive conversations with Matt and Josh, I decided to pursue an avenue of research that Matt suggested. He felt pretty confident through his  personal observations that the Palo Verde may actually do some reproduction from through cloning. The Palo Verde is a tree that has adapted well to the desert environment. It's dispersal appears to be pretty broad but it is often concentrated in washes. It specifically has adapted to growing in caliche, hardened deposits of calcium carbonate that acts like a cement holding gravel, sand, clay, and silts together in a solid mass. The Palo Verde is able to utilize voids in the caliche to develop it's root system. While very hard, this caliche layer is subject to forces of erosion. This erosion is where the fun begins with the Palo Verde. The suspicion is that when the Palo's root become exposed due to erosion that they will actually begin to grow another tree.

 This is a prime example of what I have been looking for. Notice the main tree left of center in the photo with a large root mass trailing to the right. On the right side is another tree that has matured, and between the two, new "shoots" can be seen. I think that this specimen will make a positive control.

In this picture you can see the large root heading from the main mass of trees on the right to the smaller one on the left. Is this a clone?

The same specimen from a different angle, showing that there are three separate tree systems here.

I have begun collecting samples from multiple Palo Verdes at Squaw Peak. I am collecting samples from trees I suspect to be cloning and trees that show no indications of it, all of which are in relatively close proximity to each other (map to come). Next week I will begin extracting DNA and soon hope to be running gels for analysis.

The question that looms in my head is where is the division line between one organism and another? For example in the first photo, there are three obvious tree growths connected to the same root system. Are they the same organism? Will I be able to find a situation where there are genetically identical trees that are no longer connected by the same root system? This may be difficult to do, and is definitely difficult to research. I have not been able to find literature on this subject as yet.

The digging continues...

All's fair in love and bacteria

The remaining differential test results are in and my unknown bacterium is, Staphylococcus epidermidis... or, well, maybe not...

In my last post I mentioned that there were two more tests that I was conducting. The first of these was a test referred to as the glucose O/F medium. This test would indicate wether the bacteria was capable of metabolizing carbohydrates (glucose) through oxidation or fermentation. Two test tubes of the glucose media and bromthymol blue (a pH indicator) were inoculated with samples of the bacteria. To one of these tubes a layer of mineral oil was added to create an anaerobic environment. The theory here is that a bacteria that ferments will exhibit growth in the anaerobic tube with the mineral oil, and the oxidizer will grow in the open or aerobic tube. The metabolic processes will result in the production of an acid that will change the pH indicator from dark blue to yellow. If yellow is seen in the open tube, the bacteria is an oxidizer. If the yellow is seen in the closed tube it is a fermentor.

These are the tubes after inoculation, and before incubation. Notice the layer of mineral oil on top of the media in the red capped tube.

This picture is after the incubation period as seen with backlighting. There was no perceivable difference under normal lighting. The aerobic tube on the left showed the faintest bit of a haze in the center of the tube. The anaerobic tube on the right does show what appears to be a colony growth and a yellow color change in the center of the tube. I have conducted this test in a previous experiment with a different bacteria that was a fermentor and the results were much more conclusive: the tube showed a large amount of color change. I interpreted the current result as being a weak positive for fermentation due to the apparent colony growth in the anaerobic tube. My thoughts were that maybe it was a slow grower and thus the minimal results.

I then analyzed the results of a mannitol fermentation broth. This test determines wether the fermentation of glucose results in the production of an aqueous acid or a gas. It was inconclusive on both counts. This stumped me as I believed the glucose O/F test indicated that this was a fermentor. It should have produced in the mannitol test as well, and would have been Staphylococcus epidermidis. 

I then consulted with Josh. He informed me that the glucose O/F test has been problematic in the past. My unknown should have produced in the aerobic tube and not the anaerobic tube. He did not elaborate on the reasons why the test had shown problems in the past. I suspect that the reasons are as follows. In the aerobic tube, the inoculation method required a "stab" with an inoculation needle through the gel like media. This may have created a situation that actually made it anaerobic. When the inoculation needle is withdrawn from this gel media it is plausible that the media could have "resealed" itself (the media may have been more fluid than it should have been) and not allowed oxygen to the sample once inoculated so it did not have the air needed for oxidation to occur. In the anaerobic tube, it is conceivable that the opposite occurred. Movement of the needle during inoculation could have created a void that filled with air and then was trapped in by the mineral oil. This small amount of air could have allowed some oxidation to occur and show the mild results I saw. At this point this is all speculation on my part and I need to dig deeper.

So, had the glucose O/F test showed the results it should have I would have correctly identified my unknown bacteria as Micrococcus luteus.

I'll name him George...

Last Friday I was finally able to begin some lab work!

I was issued an unknown bacterial sample (number 14) and inoculated a TSA (Trypticase Soy Agar) plate with it. The media on this "streak plate" is not selective in that it does not differentiate between different types of bacteria (most bacteria will grow on it). The streak method is a way of spreading the bacteria so that when it is allowed to incubate, individual colonies can be observed and collected for the differential tests.

This was done on Friday the 25th. The plate was left to incubate at room temperature over the weekend. On Monday morning I observed the plate and something was definitely amiss... There was bacterial growth on the plate but portions of it were missing, there were very odd small "channels" etched throughout the surface of the media, and odd specks of a white material approximately 1 to 2 mm in length.

On the right half of the plate in the above picture, the vertically oriented hazy lines are bacterial growth. The lower left section shows very little bacteria remaining and the upper left hardly any bacteria at all. Notice too, the upper left section contains many more of the small white "flecks" than other regions. I (without unsealing the plate) viewed this under a dissecting microscope at 35x magnification in hopes of determining what the contaminate on the plate could be. I immediately saw that the flecks were small wormlike organisms that were indeed moving and happily munching my bacteria! The channels I had observed were the trails left by this organism. I conferred with Matt, Josh and Robin Cotter, and we all agreed that this could possibly be Planaria, a flat worm, but it was hard to distinguish under the relatively low power of the dissecting scope.

We need more power! I observed the organism with a stronger microscope at 100x and 400x magnification.

Shown here are two examples of the organism. It is a little hard to see but the organism is segmented (definitely easier to see this under locomotion). This ruled out Planaria, and the general consensus was that it was most likely Drosophila - fruit fly larvae. At this point it was decided that this plate would remain sealed and that I would prepare another sequence of streak plates: one using the same unknown sample, one with the same unknown from a different source, and one TSA plate from the stack that I obtained the original plate from with nothing on it. This was an attempt to narrow down the possible sources of contamination. These plates were placed in the same location as the original to incubate at room temperature.

On Tuesday, the next day, I dropped in to check on the growth on the plates...

and look at that, a lone fruit fly was in the plate (that had remained sealed the entire time). I should also note that the channels I described earlier are much more easily seen in this photo. I named him George. The bacteria inoculated plates were beginning to show signs of growth, and the "blank" TSA plate showed none.

Wednesday I was able to begin differential testing. I first observed the bacterial colony morphology. 

The individual colonies were: 

Pale Yellow

Lustrous - they had a sheen of reflected light across the surface

Circular

Translucent - allowed some light to pass through but were not clear

Smooth edges

Could possibly be seen as a convex profile or umbonate profile (like a side view of a fried egg)

Next I conducted the first differential test, the Gram stain. This revealed that the bacterium was Gram +. Indicating that it has a cell wall composed of thick layers of peptidoglycan. Also during this test I could see that the individual bacterial cells were cocci - they were shaped like small round balls.

The above image is the Gram stain results as seen at 400x magnification showing the cocci shape. The purple color is the result of Crystal Violet staining the peptidoglycan in the cell walls. A Gram - bacteria would have stained pink due to the counterstain Safranin.

The next differential test to be performed was the catalase test. By adding Hydrogen Peroxide to a sample the presence of the enzyme catalase can be seen. If catalase is there, bubbles will be vigorously produced. This is the result of catalase breaking the toxic Hydrogen Peroxide down into Oxygen and water. This tells us that the bacteria can tolerate the presence of Oxygen. The unknown is catalase positive.

I did start more differential tests, but I will wait until I have results to post before describing them, and upon interpreting the results I will have a positive ID on the unknown.

On a side note, Josh, Matt and I had a very fruitful discussion about further research studies, and I am happy to say that I am very excited about it! Good things to come! I will share the topics after I conduct some preliminary research - I need to more firmly grasp the scope of what I will be doing before I try to relate it here.

Surprise!

This week I had my internship interview. Even with it being referred to as an interview, for some reason, I was under the impression that it was to be an informal "chat" concerning the topics of our research work. I look backwards and have absolutely NO IDEA where I managed to get this notion. SUPRISE! It was an interview, complete with all the fun hypothetical questions designed to see how well you can organize and relay your thoughts off the cuff. Even given my unpreparedness, I felt pretty good about it.

Just for you Josh!

After finding out that I had been officially accepted as an intern (woo, woo), I set up my schedule for lab hours, and finally found out what I will be studying for my research project. I was a little surprised to find out that I would be identifying an unknown bacterium. I had expected to see something of a more broad scope. After talking with both Matt and Josh, this makes sense in that we are in the very odd situation of starting our projects halfway through the semester and it would be very difficult to obtain good strong results from a broader subject in the limited number of weeks ahead of us. Having gone through this process before, I look forward to sharpening my techniques in the microbiology department. My head is filled with thoughts of gram stains, mannitol fermentation broth, catalase, dichotomous keys...

S-STEM Scholar in waiting...

Well, as the title suggests, I consider myself to be an S-Stem Scholar in waiting: waiting with anticipation, waiting with trepidation, waiting with excitement! I feel like I'm about to take the first step of trek into the wilderness. I say that because the peculiar ball of emotions feels the same as when I used to get dropped off in the forest knowing that I would not see anyone for days on end, not knowing what lies ahead. That is where I'm at. I have attended an orientation meeting where we discussed in generalities what I'm in for, but I can't wait to know the specifics so I can dive in! I can't wait to find out what my research topic will be. Will it be something I am familiar with, that I can build upon the knowledge I have already gained? Will it be something I know very little of, and I can gain fresh new knowledge and skills. I feel that either way I am ready for the challenge. Bring it on!

Mt Hood, Oregon: My favorite stomping grounds