The image that took me two months to obtain: A window into what it is like to do research

Most people don’t really understand what scientists actually do or what is required to do scientific research. As a result, they don’t appreciate the amount of training and work that goes into being a scientists and conducting research. I personally think that is a problem, because it leads to all sorts of confusion and misconceptions. So I want to try to clear some of that up by telling you all a bit about what I have been working on for the last few months. I hope that this example will help you to understand and appreciate just how much goes into scientific research, as well as clearing up a lot of misnomers. Many people seem to think that scientists are lazy and constantly take short cuts, falsify data, etc., but I hope that this will help you to see why that is rarely the case. I should clarify, however, that because I am going to talk about some of my own work here, there may be a few places where it sounds like I am boasting, but I assure that that isn’t the case. Quite the opposite, in fact. What I want to impress upon you is that experiences like mine are the norm in science, not the exception. I’m a 27-year-old who still carries a valid student ID. I am by no means a prodigy. At best, I’m average.

Two months to get one image

Before I can explain why it took my two months to get one image, I need to give you a bit of background about my current project. I won’t go into the details, but briefly, I am doing a microbiome project looking at how microbiomes (i.e., the bacteria, fungi, etc. that live on an organism) affect disease ecology. So I collected a bunch of samples (swabs in this case), and now I need to extract the microbial DNA from them. Once I have that DNA, I can sequence it and use those sequences to identify the species of micro-organisms that were living on my study subjects as well at looking at relative abundances.

That DNA extraction step has, however, proved quite challenging. You see, I am trying to get DNA from a range of organisms (gram negative bacteria, gram positive bacteria, and fungi), which is challenging because of differences between the groups. Gram positive bacteria, for example, have thick peptidoglycan cell walls that often require a mixture of chemicals to break them apart. In contrast, fungi have chitin cell walls that often require mechanical methods for breaking them apart (e.g., you add some tiny beads to the sample and use a “bead beater” to rapidly bash them against the sample, thus breaking apart the cells). However, not all of these methods work well together, and some methods that work really well for one group actually reduce the DNA yields from other groups. Further, to make things even worse for me, my swabs are very low yield samples, which means that there isn’t much DNA there to begin with.

This is a gel from an extraction method that completely failed. The two bright bands are control samples to ensure that the PCR and Gel worked correctly. If any of the other samples had worked, they would have shown up as similar bright bands. Thus, since my controls worked, but my samples did not, I concluded that this method does not work well for my samples.

This is a gel from an extraction method that completely failed. The two bright bands are control samples to ensure that the PCR and gel worked correctly. If any of the other samples had worked, they would have shown up as similar bright bands. Thus, since my controls worked, but my samples did not, I concluded that this method does not work well for my samples.

The result of all of this is simply that I have spent two months working in the lab trying to get an extraction method that will work reliably and consistently for all of the groups that I am interested in, even when they are very unabundant. Let me explain a bit of what that has looked like. First, I had to make a whole bunch of mock samples with known quantities of the organisms that I was interested in, that way I could test my extraction techniques on them to see if they worked. Then, I picked the method that I thought was the most promising, and I tried it on a bunch of test samples. That took a full day because there were several long steps where I had to let the samples incubate, precipitate, etc. The next day, I used a PCR (polymerase chain reaction) to try to amplify the DNA from those samples. This basically just replicates the regions of the DNA that I am interested in, that way there are enough copies for me to work with. Finally, I took those PCR products and ran them on a check gel. I won’t go into the details of what that means, but it is just a simple method for seeing wither or not you got DNA amplification, how much you have, and how long the DNA fragments are. So on a gel, if I was successful and my methods worked, I should get nice bright bands of DNA. However, I usually got gels that look like the one to the right. The only bands there are the positive controls and the ladder (you always run positives and negatives in a PCR so that you know if the it is the PCR or the extraction that failed, and the ladder is a standard that lets you tell how large the DNA fragments are).

Following that first failure, I did a bunch more reading on the method I was using, talked to several colleagues, then made some modifications to the protocol and tried again, but once again, I was met with failure. By this point, I was already four days in, but things were just getting started, because this cycle continued to repeat itself over and over again. I would try a method, it would fail, I would modify the method by changing the chemicals, temperatures, times, etc., that would fail, I would modify it again (or try an entirely different method), that would fail, so on and so forth.

This is a gel from a method that actually worked. This time, you will notice that nearly every well has a band, indicating that the extraction worked (the cells without bands were negative controls). You will also notice that some of these are smeared. That is because I was testing several methods, and I will not be using the method that produced the smears.

This is a gel from a method that actually worked. This time, you will notice that nearly every well has a band, indicating that the extraction worked (the cells without bands were negative controls). You will also notice that some of these are smeared. That is because I was testing several methods, and I will not be using the method that produced the smears.

This lasted for two months, during which, I worked in the lab at least six days a week, often working over ten hours a day trying to find a reliable extraction technique, and I want to stress that word “reliable” because it’s important. There were multiple times when I found a method that “worked” in that I could get DNA from many samples, but those methods did not give me good, consistent coverage over all of the groups of organisms that I was interested in, especially when low quantities were present. In other words, I could have done my study using one of those methods, and I would have gotten what appeared to be good results that I could easily have published, but the results would have been misleading because I didn’t use a reliable method that could consistently detect all of the groups I was interested in. Therefore, I kept trying over and over again, until finally, last week, I got the image that you see on your left. This time, you will notice that there are DNA bands, and although I have not labelled the image, I got bands even from samples that only had a handful of cells. I have also replicated these results, and they are consistent across my organisms of interest. So I finally have a reliable method, but it took me two months to get here.

Note: there are actually some issues with that gel such as smears and double bands, but that is just because I was running several methods at once. So the lanes that look messed up are from the method that I will not be using, and the lanes that look good are from the method that I will be using. There are also still a few tweaks (particularly to the PCR procedure) that I will make to clean things up a bit more before extracting the DNA from my actual samples.

The big picture: what it takes to do research

Now that you understand my suffering for the past two months, I want to talk a bit more generally about what has been required for this project. First, at the very outset I had to read a tremendous amount of scientific literature. I needed to understand the topic that I was going to study, I needed to know what other people had already found, and I needed to identify the current gaps in our knowledge. This type of background work is absolutely essential in science, and it can be quite time consuming. In many cases, this requires reading hundreds of papers.

Following that, I had to actually design a study, which means that I needed to know enough about experimental design and statistics to design a project that would let me reliably answer the questions that I was interested. So once again, a lot of background knowledge and prior training was necessary.

Simply designing a project clearly isn’t enough, however. I actually needed to do it, and for that I needed money. So, I spent several weeks writing grants to lots of different funding agencies to try to get money for this project. Several of them rejected me, but several others accepted my proposal so I got the funding. During that same time period, I also applied for research permits and filled out ethics applications so that I could actually do the project. Fortunately, all of that came together, so once I had money, permits, and ethical clearance, I immediately moved on to the next phase and actually went out into the field and collected my samples (that was also quite a challenge because it required hiking up ridiculous rainforest streams, climbing cliffs and waterfalls, etc.).

After getting the samples, I started on the lab work that I have been talking about thus far, and now that I have a working protocol, I can move on to the next stage: actually getting DNA from my real samples. Once I have the DNA from those samples, I will amplify and sequence them, and that will finally give me the actual data which I will then do a series of statistical analyses on. Following those analyses, I will write a paper on the results and submit to a journal (it will probably be another 4–6 months before I have something submitted, maybe longer).

If we put all of that together, you should start to be able to see just how much effort and how many skills are required for a project like this. I needed to have an enormous amount of background knowledge on the topic and on experimental design just to plan the study, I needed writing skills to get the grants/permits/ethics clearance, I need field skills to get the samples, I needed lab skills and a knowledge of chemistry to get the extractions working, once I finish the lab work I will need statistical skills to analyze the data, and I will need writing skills again to actually craft a paper. Perhaps most importantly though, I really need to understand the ecology of my species in order to make sense of the data that I am collecting. In other words, it’s not just enough to know a bit about the background and get the raw data, I need to actually understand things well enough that I can make sense of my results and see how they fit into the big picture. So a tremendous amount of work goes into a project like this, and a lot of knowledge and skill is required.

At this point, I again want to clarify that I’m not trying to convince you that I’m a brilliant, naturally gifted researcher, because I’m not. What I am, however, is highly trained. This brings me to perhaps my most important point in this post: science is hard, and it takes lots of training and work. I couldn’t have done a project like this when I started my scientific training almost a decade ago, and, honestly, I couldn’t even have done it a few years ago. It takes years of extremely hard work to become an academic because there is so much that you need to know. There is an overwhelming amount of knowledge that needs to be acquired and skills that need to be mastered in order to do research like this, and it takes a long time to slowly build up that knowledge and those skills. As a result, I get very upset when people who have never taken an advanced science course, never set foot in a lab, never learned statistics, etc. pretend to know more than scientists and claim that scientists are wrong on vaccines, climate change, GMOs, evolution, etc. To be clear, I’m not being elitist here. I’m not suggesting that scientists are better or smarter than everyone else, but they are highly trained and experienced, and that matters. The idea that you will understand a topic better than scientists simply by reading some websites and books is insane and extremely arrogant. Scientists are carefully trained on how to design experiments and analyze data, they spend an inordinate amount of time reading every available paper on the topics that they study, and they spend years actually doing research. That type of training, knowledge, and experience isn’t going to be toppled by a few Youtube videos.

Note: I want to be clear that I am not trying to be dismissive of citizen science projects, because I actually think that those are fantastic, largely because they help to train the public to think like scientists. Similarly, I am not trying to disparage anyone who is trying to learn about science. As I said, being a scientist is as much about training and diligence as anything else, and anyone can learn how to be a scientist. My point is simply that you shouldn’t place too much weight on your degree for Google University.

It’s a group effort

I’ve been writing this post in the first person, but I want to be totally clear that I have not been doing this alone. I have had a tremendous amount of help and support at every step of this. I have gotten help and advice from my fellow graduate students who are in the lab working on their own projects, I have gotten advice from post-doctoral researchers who recently finished their PhD’s, and I have gotten advice and help from my advisers, each of whom is an experienced researcher who spends much of their time passing on their knowledge to students. Further, this is the norm in science and has been the norm for me at every level of my training. I have had great mentors from day one and have learned a tremendous amount from working with them. Once again, that’s a type of practical learning that just can’t be matched by surfing the web. Also, you should realize that science is extremely collaborative, and most projects get input and advice from a whole host of people who don’t make it onto the actual author list. This is, in my opinion, one of the greatest strengths of science, because everyone has a different set of skills and a different knowledge base, so when those skills and knowledge bases are combined, you end up with an extraordinary and diverse amount of knowledge and ability being funneled into one project.

Most scientists aren’t incompetent or unethical

The final thing that I want to deal with in this post is the notion that scientists are either hopelessly incompetent or horribly unethical. I hear these claims all of the time, and they are ludicrous. For example, when it comes to climate change, I frequently hear people say things like, “the answer is obvious, it’s the sun, but scientists just plow forward with their beliefs instead of looking at the obvious answer” or “but the climate has changed repeatedly in the past, so it’s silly to think that the current change isn’t natural.” Claims like that are absurd. Scientists aren’t incompetent, and they have, in fact, carefully tested the natural drivers of climate change (including the sun) and consistently found that natural causes of climate change cannot explain the current warming, but our greenhouses gasses can (more details and sources here). Scientists spend a great deal of time testing every reasonable possibility that they can think of, as well as systematically refining their methods and analyses to ensure that they get accurate results. That is one of the things that I hope you will take away from my two months of trying to get a reliable extraction method working. There were lots of points that I could have stopped and used a method that I knew wasn’t accurate or reliable, but I didn’t do that because I wanted to make sure that my final results will be correct.

Once again, this type of situation is the norm for scientists. Talk to any researcher, and they will tell you very similar stories of weeks, months, or even years spent trying to get a method to work reliably, trying to refine an analysis to make it more accurate, etc. We all spend an inordinate amount of time trying to make sure that we are doing things correctly and are accounting for all reasonable possibilities. To be clear, scientists aren’t perfect, and mistakes certainly get made, but most of us care tremendously about producing high quality research because we are curious people who want answers. That’s why we went into science (it certainly wasn’t for the money, because there’s not much of that). I chose and designed my current project because it is a topic that I care about. I want my results to be correct and accurate because I want to know what the answer is. That type of curiosity is a huge driving force in science, and it forces us to be diligent.

This brings me, finally, to the topic of wide-spread corruption. Many people are under the delusion that scientists are just in it for money, and we go around falsifying data whenever it disagrees with us. That idea is, however, total nonsense. If I didn’t actually care about the results, if I was willing to falsify data, then why on earth would I spend two months working 60+ hours a week trying to get a reliable extraction method working? If I was just going to make up the results, then I could have just gone ahead with one of the crappy, unreliable methods. For that matter, I could have just skipped the lab work altogether and sat down at my compute and generated fake results. Similarly, the idea that I would get to the end of this project only to have someone pay me to keep the results quiet or to change the results is nuts. Look, by the time that it is all said and done, I will have invested close to a year of my life into this project. I have poured everything into it, and there is no way on earth that I am going to get to the end and either not publish or change my results just because someone offers me a few thousand dollars. I am not so provincial and neither are the majority of scientists. Again, we don’t do this for the money. If money was what we cared about, we would have chosen a different field. We do this because we want to know how things work. We do this because we care about expanding mankind’s knowledge and making the world a better place, and the vast majority of us would not be willing to bury research for a simple bribe. To be clear, there are bad apples in every group, but again, what I am describing here is not out of the ordinary. Go talk to any scientists anywhere in the world and they will have similar stories, because this is the norm for science.

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3 Responses to The image that took me two months to obtain: A window into what it is like to do research

  1. Philipp says:

    Sounds like the common everyday struggle to me. Agree both with your experience and comments. I do not think scientists are above criticism by any means, yet how easily people discard our critical statements is nerve-wracking. It is a negation of our struggle for truth without basis, the epitome of ignorance.

    “Nevertheless, (Thomas Jefferson) believed that the habit of skepticism is an essential prerequisite for responsible citizenship. He argued that the cost of education is trivial compared to the cost of ignorance, of leaving government to the wolves. He taught that the country is safe only when the people rule.”
    ― Carl Sagan, The Demon-Haunted World: Science as a Candle in the Dark


  2. Peter says:

    Well written. It captures well the time and effort we put into scientific research because, as you say, “we want to know how things work”. I think a value of citizen science is that it can give the general community a small taste and better appreciation of what professional scientists do.


  3. foodnstuff says:

    Very good post. I didn’t finish my degree, but it’s easy to accept everything you’ve said. Science and the scientific method should be taught from day one in school. Then at least people will understand even if they don’t take up science as a career.


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