The Road to Research #3: So you want to use laboratory equipment?

Okay, so now that you’ve developed a research question it’s time to start preforming your experiments. But, before you can begin to collect data you have to understand how to use the scientific instruments necessary. Some experiments require simplistic technology: a caliper or a scale, where as other experiments demand more advanced tools: a million dollar microscope, or a surprisingly expensive solvent. In the summer of my sophomore year my technician skills were put to the test when I applied for the CURE grant at CU Boulder and began working in the Infectious Disease Unit at the Anschutz Campus. The Clinical Undergraduate Research Experience (CURE) research grant allows students to work in clinical laboratories and experiment with a career in the medical field. I was fortunate enough to receive this $2,500 grant and work under Dr. Elizabeth Connick who is a HIV researcher and infectious disease practitioner. When you begin your research career you quickly learn that each laboratory group has their own ‘culture’. This is usually due to the leadership style of the PI, the amount of lab members, and of course the facilities available. When I began doing clinical research in the Connick Lab I got to experience a different type of research culture as well as alternate technology. There are three main categories of equipment you will find in a laboratory and you will have to use in order to answer your research question: instruments, reagents, and software.

#1: The Instruments of Science!

The Microscope:

Welcome to the land of microscopes, a surprisingly diverse family of observational tools. Microscopes are awesome, they have allowed scientists for centuries at the smallest objects in nature. Be careful, the microscope can get addicting!

never_do_this

XKCD- ‘Never Do This’

There are three main categories of microscope: optical, fluorescence, and electron, you will most likely be working with the first two (unless you work for Reed Richards). The optical node of the tree has two different types of microscopes: a simple and a compound scope. The most obvious difference between these two types is eye piece number (1 vs 2), however, there are even more differences. For example, the simple microscope is often used for dissection of ‘big’ objects, it has only one objective and its magnification power is limited, this type of microscope is ideal for examining the stomach contents of a shrimp (see picture below). Compound scopes on the other hand have multiple objectives and magnification ranges and are ideal at looking at mitosis in plant cells. Fluorescence microscopes (or sometimes called confocal microscopes) are usually used to examine structure within the cells, for example, this is the type of microscope I used in order to examine the HIV virus in lymphatic tissue at Anschutz. This type of microscope allows you to tag certain proteins on different organelles with fluorescent markers and examine them using laser light. Without using a fluorescent microscope one would not be able to see the colors assigned to the cellular structure. For example, in the picture bellow the proteins of the nucleus have been tagged with blue color, the cell walls with red, and the cytoplasm with green. Electron microscopes are pretty complicated, so I’ll just say a little about them. Using an EM requires one to cover the sample with gold and inundate it with electrons, the electrons will bounce off which will give the user a clear picture of the most microscopic of objects (the picture in the example are viruses!).

micro

 

Every microscope is different, and learning how to use one always requires a learning curve. My advice is to remember that most microscopes cost hundreds of thousands of dollars, but also to maintain a sense of confidence, it’s fairly hard to break a microscope. Once you’ve developed some mad skillz you can get some truly amazing shots, here are some I’ve gotten over the years.

8506701_orig1

Pygidiopsoides sp. found in the testes of a snail, a simple dissection microscope was used.

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HIV infected lymph node tissue, the green cells are double positive cells, a florescent microscope was used.

11079645_10205245162542874_938451888857428804_n

Leishmania donovani, this parasite causes visceral leishmaniasism (black fever), a compound microscope was used.

For more information about how to use microscopes check out my favorite site: http://www.cas.miamioh.edu/mbiws/microscopes/index.html

Other Instruments used for Measurement:

Mass:

  • Analytical balance
  • Triple beam balance

Distance:

  • Caliper
  • Ruler

Volume:

  • Pipettes
  • Pipette Men
  • Graduated Cylinder
  • Beakers
  • Volumetric Flasks
  • Syringes
  • Buret

#2: Serums, Reagents, and Enzymes

tests

XKCD- ‘Tests’

At some point in your experiment you will have to make or use reagents, serums, and enzymes, these solutions are often used to induce a chemical reaction necessary to the experiments success. There are thousands, probably millions, of solutions, but there are some that are more universal throughout laboratories. For example, I’ve used everything from ethanol for cleaning dissection tools to rare and expensive antibodies for indirect tagging of cellular structures. Instead of trying to cover some of the most common solutions, I will instead outline important rules to remember while using and making these reagents.

  1. Get permission from a laboratory manager or a PI before you begin making or using any solutions.
  2. Make a list of all of the serums/reagents/enzymes you will need for your experiment.
  3. Find ‘recipes’ and protocols for each of these solutions from published papers, google, or even your lab mates.
  4. Gather all of your ingredients and ensure you have enough in every bottle. You may need to order more before you can begin creating reagents.
  5. Ensure to do all your calculations before beginning anything, after you’ve done them, check them. And then check them again. And then have someone else check them. I can’t tell you how many times I’ve wasted an entire afternoon due to a misplaced decimal point.
  6. For safety’s sake be sure to read all warnings on any bottles, do research on the chemicals you are using, and always take precautions in the form of gloves, masks, and goggles.

List of common chemicals and their hazards:

  • Acetone- flammable
  • Agar – not hazardous
  • Antibodies- potential biohazard
  • Bovine Serum Albumin (BSA)- irritant
  • Cell culture media- not hazardous (you could drink this… theoretically…)
  • Crystal Violet- not hazardous
  • DSMO reagent- irritant and flammable
  • Ethanol- irritant and flammable
  • Formaldehyde- poisonous
  • Immersion oil- not hazardous (salad dressing anyone?)
  • Liquid nitrogen- hazardous
  • Methanol- irritant, flammable, poison
  • Restriction enzymes- not hazardous
  • TBS Buffer- not hazardous
  • Triton X-100- irritant

 

#3: Software and Programs

Thankfully you are doing science in the modern age, this means that there are many types of software and programs designed to help you collect massive amounts of data, recognize seemingly random patterns, and ultimately organize your results into something noteworthy. One of the most tedious parts of most experiments is counting and measuring, especially when your subjects are cells or parasites and there are millions of them in each sample. You could spend an entire Saturday hunched over a microscope with a counter clicking away, or you could take a picture, upload it into a program and let the software recognize the objects and interest and tally them for you in minutes (CellCounter or CellProfiler are great programs for this). Sometimes you have to measure a cells diameter, or it’s cytoplasm to nucleus ratio, and you can create your own programs to do this. In fact, while working in Dr. Connick’s lab I worked on improving a very simple computer program built specifically for lymphatic cells. All of these types of programs allow a researcher to collect large amounts of data in a very quick amount of time and it limits the potential ‘human error’ of missing certain objects (something that’s easy to do when falling asleep at the bench). Once you have this data computer programs (like R, Qiime, or other pipelines) can help you filter your data for quality, sort it in a way that answers your research question, and create figures for you to convey your results to others.

If microscopy requires a learning curve, then these programs represent a learning asymptote. They are extremely difficult to learn and can be quite frustrating at times, but once you are able to work out the bugs they should (theoretically) make your life easier as a researcher.

automation

XKCD- ‘Automation’

This is a very basic overview obviously, it’s impossible to thoroughly describe all the scientific instruments I’ve used or you may use in 2,000 words or less. However, if I didn’t cover something you needed (or if you’re a lab nerd) I really recommend reading the book ‘At the Bench’ by Kathy Barker.

Check out my next blog entry where I’ll be discussing my experience getting the UROP research grant and how to present your research in a paper, a poster, and a presentation.

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