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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: bmartinezvaz01@hamline.edu.
    • ©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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3. Hunt S1997Measurement of nitrogenase activity in N2-fixing nodules of soybeanPresented at the 18th Conference/Workshop of the Association for Biology Laboratory Education (ABLE)Boston University
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5. Janssen P2006Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genesAppl Environ Microbiol721719172810.1128/AEM.72.3.1719-1728.2006165176151393246 http://dx.doi.org/10.1128/AEM.72.3.1719-1728.2006
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9. Rinaudi L, Isola MC, Giordano W2009The ribosomal RNA is a useful marker to visualize rhizobia interacting with legume plantsBiochem Mol Biol Educ3218719010.1002/bmb.2004.494032030353 http://dx.doi.org/10.1002/bmb.2004.494032030353
10. Rosenzweig JA, Jejelowo O2011What microbes are lurking in your investigation house? Identification of unknown microorganisms using a PCR-based lab experimentAm Biol Teach7333133510.1525/abt.2011.73.6.5 http://dx.doi.org/10.1525/abt.2011.73.6.5
11. Sadowsky MJ, Keyser HH, Bohlool B1983Biochemical characterization of fast- and slow-growing rhizobia and nodulate soybeansIntl J System Bacteriol3371672210.1099/00207713-33-4-716 http://dx.doi.org/10.1099/00207713-33-4-716
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/content/journal/jmbe/10.1128/jmbe.v16i2.973
2015-12-01
2017-09-25

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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