1887

An Alternative Approach to “Identification of Unknowns”: Designing a Protocol to Verify the Identities of Nitrogen Fixing Bacteria

    Authors: Betsy M. Martinez-Vaz1,*, Roxanne Denny2, Nevin D. Young2, Michael J. Sadowsky3
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    Affiliations: 1: Department of Biology, Hamline University, Saint Paul, MN 55104; 2: Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108; 3: Department of Soil, Water and Climate, University of Minnesota, Saint Paul, MN 55108
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Supplemental materials available at http://jmbe.asm.org
    • *Corresponding author. Mailing address: Department of Biology, Hamline University, 1536 Hewitt Ave., Saint Paul, MN, 55104. Phone: 651-523-2493. Fax: 651-523-2620. E-mail: [email protected].
    • ©2015 Author(s). Published by the American Society for Microbiology.
    Source: J. Microbiol. Biol. Educ. December 2015 vol. 16 no. 2 247-253. doi:10.1128/jmbe.v16i2.973
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    Abstract:

    Microbiology courses often include a laboratory activity on the identification of unknown microbes. This activity consists of providing students with microbial cultures and running biochemical assays to identify the organisms. This approach lacks molecular techniques such as sequencing of genes encoding 16S rRNA, which is currently the method of choice for identification of unknown bacteria. A laboratory activity was developed to teach students how to identify microorganisms using 16S rRNA polymerase chain reaction (PCR) and validate microbial identities using biochemical techniques. We hypothesized that designing an experimental protocol to confirm the identity of a bacterium would improve students’ knowledge of microbial identification techniques and the physiological characteristics of bacterial species. Nitrogen-fixing bacteria were isolated from the root nodules of and prepared for 16S rRNA PCR analysis. Once DNA sequencing revealed the identity of the organisms, the students designed experimental protocols to verify the identity of rhizobia. An assessment was conducted by analyzing pre- and posttest scores and by grading students’ verification protocols and presentations. Posttest scores were higher than pretest scores at or below p = 0.001. Normalized learning gains (G) showed an improvement of students’ knowledge of microbial identification methods (LO4, G = 0.46), biochemical properties of nitrogen-fixing bacteria (LO3, G = 0.45), and the events leading to the establishment of nitrogen-fixing symbioses (LO1&2, G = 0.51, G = 0.37). An evaluation of verification protocols also showed significant improvement with a p value of less than 0.001.

References & Citations

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2. Emmert EB 2013 Biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationale J Microbiol Biol Educ 14 78 83 10.1128/jmbe.v14i1.531 23858356 3706168 http://dx.doi.org/10.1128/jmbe.v14i1.531
3. Hunt S 1997 Measurement of nitrogenase activity in N 2-fixing nodules of soybean Presented at the 18th Conference/Workshop of the Association for Biology Laboratory Education (ABLE) Boston University
4. Janda JM, Abbott SL 2007 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls J Clin Microbiol 45 2761 2764 10.1128/JCM.01228-07 17626177 2045242 http://dx.doi.org/10.1128/JCM.01228-07
5. Janssen P 2006 Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes Appl Environ Microbiol 72 1719 1728 10.1128/AEM.72.3.1719-1728.2006 16517615 1393246 http://dx.doi.org/10.1128/AEM.72.3.1719-1728.2006
6. Kneen B, La Rue TA 1983 Congo red absorption by Rhizobium leguminosarum Appl Environ Microbiol 45 340 342 16346177 242283
7. Olsen G, Woese CR 1993 Ribosomal RNA: a key to phylogeny FASEB J 7 113 123 8422957
8. Ponder MA, Sunmer S 2009 Use of case studies to introduce undergraduate students to principles of food microbiology, molecular biology, and epidemiology of food-borne disease Biochem Mol Biol Educ 37 156 163 10.1002/bmb.20277 21567725 http://dx.doi.org/10.1002/bmb.20277
9. Rinaudi L, Isola MC, Giordano W 2009 The ribosomal RNA is a useful marker to visualize rhizobia interacting with legume plants Biochem Mol Biol Educ 32 187 190 10.1002/bmb.2004.494032030353 http://dx.doi.org/10.1002/bmb.2004.494032030353
10. Rosenzweig JA, Jejelowo O 2011 What microbes are lurking in your investigation house? Identification of unknown microorganisms using a PCR-based lab experiment Am Biol Teach 73 331 335 10.1525/abt.2011.73.6.5 http://dx.doi.org/10.1525/abt.2011.73.6.5
11. Sadowsky MJ, Keyser HH, Bohlool B 1983 Biochemical characterization of fast- and slow-growing rhizobia and nodulate soybeans Intl J System Bacteriol 33 716 722 10.1099/00207713-33-4-716 http://dx.doi.org/10.1099/00207713-33-4-716
12. Science Learning Hub 2013 Student activity: clover and nitrogen fixation http://sciencelearn.org.nz/ University of Waikato New Zealand
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14. Somasegaran P, Hoben HJ 1994 Handbook for rhizobia: methods in legume rhizobium technology Springer-Verlag New York 10.1007/978-1-4613-8375-8 http://dx.doi.org/10.1007/978-1-4613-8375-8
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2015-12-01
2019-08-18

Abstract:

Microbiology courses often include a laboratory activity on the identification of unknown microbes. This activity consists of providing students with microbial cultures and running biochemical assays to identify the organisms. This approach lacks molecular techniques such as sequencing of genes encoding 16S rRNA, which is currently the method of choice for identification of unknown bacteria. A laboratory activity was developed to teach students how to identify microorganisms using 16S rRNA polymerase chain reaction (PCR) and validate microbial identities using biochemical techniques. We hypothesized that designing an experimental protocol to confirm the identity of a bacterium would improve students’ knowledge of microbial identification techniques and the physiological characteristics of bacterial species. Nitrogen-fixing bacteria were isolated from the root nodules of and prepared for 16S rRNA PCR analysis. Once DNA sequencing revealed the identity of the organisms, the students designed experimental protocols to verify the identity of rhizobia. An assessment was conducted by analyzing pre- and posttest scores and by grading students’ verification protocols and presentations. Posttest scores were higher than pretest scores at or below p = 0.001. Normalized learning gains (G) showed an improvement of students’ knowledge of microbial identification methods (LO4, G = 0.46), biochemical properties of nitrogen-fixing bacteria (LO3, G = 0.45), and the events leading to the establishment of nitrogen-fixing symbioses (LO1&2, G = 0.51, G = 0.37). An evaluation of verification protocols also showed significant improvement with a p value of less than 0.001.

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FIGURE 1

Examples of identification protocols proposed by microbiology students to verify the identity of rhizobia isolated from root nodules.

Source: J. Microbiol. Biol. Educ. December 2015 vol. 16 no. 2 247-253. doi:10.1128/jmbe.v16i2.973
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