Microbe Monday: A sterile stick and other tools

My previous advisor used to joke that all you need to do microbiology is a sterile wooden stick. You can use this simple tool too isolate colonies, pick colonies, inoculate media and isolate DNA*. If you were to visit my bench, you would find a bucket of sterile sticks as I use them almost every day. However, I need a lot more than a wooden stick to get my research done. The machines I use most often are the thermocycler, FPLC, biacore and any kind of spectrophotometer. Unfortunately, my favorite tool, the microscope, is one I rarely use.

One of the major reasons I love this instrument because it enables me to actually see the microbes I am studying. Since my work typically involves using RNA, DNA or protein, I spend most of my time looking at tubes containing a clear solution, or a if I'm lucky, a solution containing a dye. If not a tube, then I'm probably viewing a stained gel (acrylamide or agarose). It's just not as visually stimulating as looking at something under the microscope.**

Another reason I like to use the microscope is because you can take pictures or video of what you observe. Pictures and videos typically go over really well in a seminar and when applicable, they are great in a paper. The presence of images or video in a paper was often a deciding factor when I was deciding on a paper for journal club. I think everyone agrees that it's nice to see something besides a graph or table every now and then.***

For researchers like me, there just isn't usually a reason to have photos or video from a microscope. However, when I can, I choose assays that utilize a microscope. For example, to assess translation, I'll use a reporter gene like GFP so that not only do I get to use the microscope, but I get to look at fluorescence and that is way cool. I've also used fluorescence to assess protein-protein interactions. In both of these cases I can use the microscope for qualitative "yes or no"-type of data and then if need be, I can use a fluorometer for more quantitative data.

To illustrate my point, here are images from wikimedia commons. I wanted to include videos, but blogger wasn't cooperating.****

Original figure legend: Multiple fluorescence 2PE imaging. 2PE multiple fluorescence image from a 16 μm cryostat section of mouse intestine stained with a combination of fluorescent stains (F-24631, Molecular Probes). Alexa Fluor 350 wheat germ agglutinin, a blue-fluorescent lectin, was used to stain the mucus of goblet cells. The filamentous actin prevalent in the brush border was stained with red-fluorescent Alexa Flu or 568 phalloidin. Finally, the nuclei were stained with SYTOX ® Green nucleic acid stain (2).

Polymicrobic biofilm grown on a stainless steel surface in a laboratory potable water biofilm reactor for 14 days, then stained with 4,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy (3).

*Once the DNA is precipitated, you can wind it around a sterile stick or toothpick in leu of centrifugation. This is not really effective if you aren't isolating a large quantity of DNA.

** Yes, you can visualize your data in ways that don't involve a microscope and as a general rule it's awesome to obtain data, be that data in the form of a band, a sensogram or absorbance reading, but in my opinion seeing your data in a living organism is way cool.

***Not that anything is wrong with a table or graph. Pretty much all my data is presented in this form.

****I know that there are many compelling images and videos utilizing fluorescence microscopy, but I am not sure what the rules are regarding image and video usage from scientific papers. If anyone would like to enlighten me, please feel free. If anyone knows of some better images, that I can post without committing any kind of offense or spending money, please let me know and I will be happy to post them.

(2) Multi-photon excitation microscopy. BioMedical Engineering OnLine, 2006, 5:36.DOI:10.1186/1475-925X-5-36.

(3) Centers for Disease Control and Prevention Rodney M. Donlan: "Biofilms: Microbial Life on Surfaces"

Get over yourself.

Rising to the challenge. That's the topic of this month's Scientiae Carnival, as put forward by Candid Engineer.
At first I wasn't sure what to write about because as a scientist, I feel like I face challenges almost everyday. Granted, they aren't all huge or difficult to overcome, but more than enough of them are. While contemplating the topic, I ran across a post by Sciencewomen about her MS experience, and inspiration hit. I'll write about one of my biggest, and unfortunately on-going, challenges: myself.
Like many people I possess qualities that are self-destructive. Particularly, I hold a grudge, I am quick to judge and bitterness and resentment come a little too easily. Making it through my masters program brought all of these qualities to the surface and in doing so, it also made me realize that I needed to change.
In the beginning things were fine, even though I applied for the Ph.D. program and was offered the M.S. program as a consolation prize. Since my undergraduate degree was not in science, math or anything analytical, I thought this decision by the admissions committee was understandable. I decided that since I wanted to complete the Ph.D. program, I would just enter the M.S. program but approach it the same way as a Ph.D. student, taking all the same classes, etc. The first half of the fall semester, proved difficult, but then I hit my stride. I started doing well in classes and in the lab and I started to realize that there was really no significant difference between the Ph.D. students and myself. The faculty in the department noticed this too and inquired with the graduate school about transferring me into the Ph.D. program. Unfortunately, the graduate school would NOT budge on this. They were worried that this would start a trend where undeserving M.S. students entered the Ph.D. program through the “back door.”
FINE! What. Ever.
Over the next few years, my fellow Ph.D. students took their qualifying exams and became Ph.D. candidates while I toiled away on a shitty project that was going absolutely nowhere, all the while getting more and more frustrated. What finally broke the barrier from frustration to out and out bitterness and resentment was: (1) figuring out that the graduate school was more than happy to bend plenty of rules, and (2) understanding that the amount of work required to get a M.S. varied wildly from student to student.
The graduate school bent rules for everyone, everyone but me it seemed. For example, students who were making horrible grades were given probationary period after probationary period even thought the rules clearly stated that they should have been kicked out. Furthermore rules regarding time frames for qualifying exams and graduation were routinely ignored. I didn’t particularly care if the graduate school was flexible on these points, but I felt that if one rule could be bent (and in some cases totally ignored), then why couldn’t all of them. If you aren’t going to follow the rules, then get rid of them.
In addition to feeling like I was the only student in the school that had to follow any rules, I also felt like I was being held up to a much higher standard than the other M.S. students. (Granted, there was only one other M.S. student in my department, but bitter people don’t really care about the statistical significance of their sample size.) The incident that finally pushed me over the edge was finding out that the M.S. student (who worked in the lab of one of my committee members) who started the program 2 years after I did, was going to graduate one semester after me. Not because they had enough data, or an awesome project that went smoothly, but because they threatened to quit the program. They claimed they were having a nervous break down and just couldn’t take it anymore. What was the solution? Offer the M.S. degree in exchange for one more semesters worth of work.
I was fucking livid when I found this out. This person's PI was on my committee, and they were also actively campaigning for me to put in yet another semester of work a mere two weeks after the incident with their own M.S. student. I refused and thankfully the rest of the committee felt it was unnecessary. Thankfully, I graduated at the end of the semester.
Once I finished the M.S., I went straight into the Ph.D. program and took my qualifying exam within a year in an effort to "catch-up." I tried to leave the M.S. experience behind, but I couldn't. Every time I heard about rules getting bent for a student or encountered Ph.D. students without the ability to learn or think about science, I would feel the bitterness and resentment rising up inside, making me want to explode. To make matters worse, I dwelled on the fact that I was behind everyone else.
I was miserable, but why? I was in the Ph.D. program and doing well, so what the fuck was my problem? I was the problem. Holding onto all of those negative feelings and constantly reminding myself of every misfortune that I encountered was making me unhappy and I needed to stop.
Since then, I try to take things with the proverbial grain of salt. I also try very hard not to complain about unfair situations because when I complain, I get too upset and too caught up in the negativity and, as a result, all the self destructive feelings rise to the surface. I am by no means perfect. I still complain, but I try to avoid carry things around with me the way that I did in the past and I am finding that I am a lot happier.
So, my M.S. taught me that I needed to get over myself. I needed to change my behavior before it took over my life and made me a miserable person that no one wanted to spend time with or work with. This realization was the most important thing that came out of my M.S. program.
Changing your behavior is one of the most difficult challenges in life. It takes constant work and self critique and unfortunately the progress is painfully slow, but it is rewarding when you find success.

Ch-ch-ch-ch-changes

My first degree was essentially a communications degree with an emphasis in public relations. I parlayed this degree into my first job, a fundraiser for the Muscular Dystrophy Association. It was fun and at the end of the day, it was nice to know that I helped someone. Unfortunately, once I learned how to do the job, I got bored...really, really bored. I considered finding a new job, but I figured it would end the same way. I needed to change career fields, not just jobs. This lead to two important realizations about myself:

1. I find many topics interesting, so no matter what I majored in, I probably would have thought it was the right career for me until I took a job in that field.
2. No matter how much I didn't want to admit it before, I was better at analytical subjects like math and science.

Ultimately, I decided that a career in science or health care was exactly what I needed. Why? Let's revisit numbers 1 and 2 from above.
Like I stated above, I like to learn and I find many things interesting, but I couldn't very well go back to school, major in something new, enjoy it while learning about it and then find out again that the actual application of the new degree was boring. I needed the job itself to require or consist of learning so that I could avoid the boredom. So, I thought about what careers would require constant learning while allowing me to analyze the crap out of things. The answer was science.

Now for the transition.
I went back to school and started taking prerequisites for graduate school. Unfortunately, this required moving back in with my parents for a year so that I could take more classes at a time and get into graduate school faster. This was difficult for me, but not because of my parents. They were pretty excited about it, but it embarrassed me terribly. I swore to myself and my parents to never, no matter what, move back home after I went to college.
Everyone in my classes were a couple of years younger than me and I wasted a lot of time and energy feeling like a loser because of this. Since I started school early, I was accustomed to being the youngest. Now, I felt like a loser that lived with their parents who was behind. Not a feeling I relished.
When I finally started graduate school, things didn't exactly change. I did not actually obtain a degree in science. I only took the prerequisites and even though I made good grades in those required courses, my learning curve at the beginning of graduate school was steep as hell. Right away I enjoyed two more new experiences: studying and near-failing. I scored the second lowest grade on my first graduate school exam. Studying and making low grades...wtf? There were even rumors that faculty thought I wouldn't make it, but I did.

In the end, it only took that fist semester to get up to speed. Thank goodness. Later, I passed my candidacy exam unconditionally and a committee member informed me that it was the best oral exam he had ever witnessed. Not too bad for a communications major with no science degree that nearly failed out of graduate school in semester one.

Despite how well everything turned out, I still view this as a difficult transitional time in my life and lately I find myself thinking about it because I am nearing the end of my graduate career and moving to a new lab that studies microbes, but not in the same way graduate school lab (GSL) studies them. Post-doc lab is heavy on the biochemistry and structure while GSL is into gene regulation. I look forward to learning new techniques and I know that when it is over I will be an extremely well-rounded microbiologist, but I am a little intimidated by all the new equipment.

I think it is going to be fine. My past experience tells me so.