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2.2: Using the spectrophotometer to evaluate your pipetting skills - Biology


Since you will be using micropipettes for all of your experiments, the quality of your results will depend on proper operation of the micropipette. Your results will also provide information about whether the pipettes are functioning properly.

In these exercises, you will be using the spectrophotometer to determine if your pipetting is accurate and precise. You will
be using micropipettes to combine various volumes of water and solutions of a blue dye, bromophenol blue (BPB). You will measure the absorbance of the resulting solutions at 590 nm (A590), which is close to the absorbance maximum of bromophenol blue at neutral pH. Measuring errors will be reflected in the spectrophotometer readings.

The spectrophotometer readings provide an indirect measurement of pipette performance. The proper way to calibrate the micropipettes would be to weigh out volumes of water, which
has a specific gravity of 1.0 g/mL. Unfortunately, we do not have enough balances with sufficient accuracy for the class to perform the measurements. If you suspect inaccuracies in the micropipettes that you are using, refer them to the teaching staff, who will test them properly.


Applied spectrophotometry: Analysis of a biochemical mixture

Spectrophotometric analysis is essential for determining biomolecule concentration of a solution and is employed ubiquitously in biochemistry and molecular biology. The application of the Beer-Lambert-Bouguer Lawis routinely used to determine the concentration of DNA, RNA or protein. There is however a significant difference in determining the concentration of a given species (RNA, DNA, protein) in isolation (a contrived circumstance) as opposed to determining that concentration in the presence of other species (a more realistic situation). To present the student with a more realistic laboratory experience and also to fill a hole that we believe exists in student experience prior to reaching a biochemistry course, we have devised a three week laboratory experience designed so that students learn to: connect laboratory practice with theory, apply the Beer-Lambert-Bougert Law to biochemical analyses, demonstrate the utility and limitations of example quantitative colorimetric assays, demonstrate the utility and limitations of UV analyses for biomolecules, develop strategies for analysis of a solution of unknown biomolecular composition, use digital micropipettors to make accurate and precise measurements, and apply graphing software. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(4):242–250, 2013


Introduction

The biology department at Chestnut Hill College has endeavored to incorporate the research process into all of its undergraduate biology courses, beginning long before the recommendations in Vision and Change in Undergraduate Biology Education (AAAS, 2011). In most undergraduate microbiology courses, the laboratory portion is very technique oriented (e.g., Gram staining, identification of bacteria, testing for antimicrobial resistances). While all these concepts and techniques are important to teach, they do not necessarily utilize the research process. The challenge of teaching in the sciences is not only conveying knowledge in the discipline, but also developing essential critical thinking, data analysis, and scientific writing skills. Therefore, I have designed a laboratory exercise that teaches the simple concepts of how bacteria grow and how bacterial growth can be measured, while also emphasizing the development of a hypothesis, research design, data analysis, and the writing of a research paper (see Table 1 for overview).

Overview of a laboratory exercise on bacterial growth.

First Draft and Final Draft

Escherichia coli is an ideal microorganism for undergraduate research projects. It has a simple, completely sequenced genome and a rapid growth rate under optimal conditions is easy to handle and cultivate and is relatively harmless (Zimmer, 2008). Therefore, in a 3-hour laboratory session, when an overnight culture of E. coli is heavily inoculated into media already at the appropriate temperatures, it is possible to obtain a complete growth curve. (My PowerPoint presentation for this lab is available at https://mckernanmicrobiology.wikispaces.com.)

The objectives of the laboratory exercise are as follows:

To review and practice scientific inquiry. I walk the class through the steps of scientific inquiry. We discuss the terms “hypothesis” and “theory” the difference between “discovery science” and “hypothetico-deductive science” the importance of a clearly defined purpose for a research study and the effect of culture conditions on bacterial growth and the importance of studying bacterial growth.

To use spectrophotometry and standard plate method to determine the doubling time of E. coli during the exponential phase of growth. Most students have taken a chemistry course and used spectrometry to measure pigmented solutions, but here bacteria are measured as particles in solution. As they take optical density readings, they very quickly see a growth curve developing. The standard plate method invites review of serial dilutions, the concept of colony-forming units, and how to determine the concentration of microorganisms. Using two methods gives the students an opportunity to discuss how different methods can be used to measure the same parameter – in this case, doubling time. But it is also important to know what the different methods really measure and how that might affect interpretation of the data, and that there are advantages and disadvantages of methods.

To use Excel in graphing and calculating doubling time. The concepts of independent and dependent variables and the construction of tables and graphs are reviewed. Most students at this level have used Excel to make tables and graph data. But changing the y-axis to a logarithmic scale, adding an exponential line, and using the equation for the line to calculate doubling time is new to many students. Analyzing the raw data, optical density readings, and colony forming units/mL to determine doubling time is challenging and a highlight of this laboratory exercise.

To write a science research paper. The parts of a scientific paper are covered in detail (see https://mckernanmicrobiology.wikispaces.com) descriptions of what should be included in each section are posted for students to use as a guide in writing the paper. Also covered in detail are presentation of data in tables and graphs and the calculations for determining doubling time. Students submit a first draft, edit it, and turn in a final draft for grading.

I have taught this laboratory exercise for 6 years. The classes are usually composed of biology majors in their junior year, with an occasional sophomore or a few seniors. The students have always enjoyed it, and they frequently say in evaluations that it was the best lab of the semester. Students have often said that they like the lab because of its hands-on aspects: taking readings, seeing the growth curve emerge, pipetting, serial dilutions and plating, and the quantitative analysis of raw data to calculate doubling times. This exercise is usually run in the fourth week of the semester, so that students already have some practice with handling of bacterial cultures and aseptic technique. In the lecture portion of the course, the concepts of binary fission, four phases of bacterial growth, factors affecting bacterial growth, and techniques for measuring bacterial growth are covered. Therefore, the two techniques used to measure bacterial growth in this experiment – optical density measurement and standard plate method – have already been discussed.

In the first laboratory session, the process of scientific inquiry and the importance of studying bacterial growth are discussed. I lead the discussion toward why it might be important to study the effect of temperature on bacterial growth, and a hypothesis is developed for the growth of E. coli at 37°C and 25°C. The techniques of spectrophotometry and serial plate dilutions for measuring the growth rate of bacteria are established methodology for the microbiologists. It is also well documented that E. coli have a doubling time of 15–20 minutes under optimal conditions. I emphasize that an understanding of how this number was arrived at is important. It is necessary to determine what the doubling time is under our particular growth conditions. Also, science needs to be repeatable. Can we repeat the results observed by others? Why would we need to know that? What are optimal conditions? What if we wanted to determine a better growth medium for improved growth rates? We chose 37°C because it is human body temperature and E. coli is part of the normal flora of the gut and 25°C because it is room temperature but other temperatures could have been selected.

Next, the procedures are reviewed. The students inoculate their trypticase soy broth (TSB) tubes and learn how to use the spectrophotometer to measure optical density, which includes setting the wavelength, zeroing the instrument with an uninoculated broth tube, and then measuring the optical density of their freshly inoculated TSB tube. This measurement is their T = 0 minutes reading, and the experiment has begun. Using the standard plate method, two 1-mL samples are removed at two different time points in the experiment.

In the second session, “Parts of a Scientific Paper” are presented in a PowerPoint (https://mckernanmicrobiology.wikispaces.com). Then, as a class, the growth-curve graphs that students prepared from OD data collected in session 1 are evaluated. The students have varying degrees of expertise in Excel, and I have found that the majority of the class needs help with formatting their graphs. The data from the standard plate method are collected, and colony-forming units (CFU)/mL are calculated. An explanation of how doubling time is calculated from each method is covered (https://mckernan microbiology.wikispaces.com). Then the results are evaluated and the class brainstorms on whether the results confirm their hypothesis, and why or why not whether the two methods gave comparable doubling times, and why or why not the advantages and disadvantages of the two methods and finally, how the procedures might be improved and what further study might include. Students find that the most difficult section of a scientific paper to write is the conclusion section, so I cover again what should be included in that section:

Summary of the doubling times at the two temperatures and with both methods.

Did you reach conclusions about the initial hypotheses?

Are your results comparable to the conclusions of others?

Are the two methods comparable? Why or why not?

Identify sources of error and basic inadequacies of techniques.

What improvements of methods and further steps are needed in research on the problem?


Watch the video: Spectrophotometer (November 2021).