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Isolation and Characterization of Strains from Soil: A Laboratory Capstone Experience

    Authors: Kim R. Finer1,*, Lee Fox2, John J. Finer3
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    Affiliations: 1: Department of Biological Sciences, Kent State University at Stark, N. Canton, OH 44720; 2: Department of Psychology, Kent State University at Stark, N. Canton, OH 44720; 3: Department of Horticulture and Crop Science, The Ohio State University, Wooster, OH 44691
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 02 December 2016
    • ©2016 Author(s). Published by the American Society for Microbiology.
    • [open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.

    • Supplemental materials available at http://asmscience.org/jmbe
    • *Corresponding author. Mailing address: 6000 Frank Ave. N.W., N. Canton, OH 44720. Phone: 330-244-3398. Fax: 330-494-6121. E-mail: kfiner@kent.edu.
    Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 444-450. doi:10.1128/jmbe.v17i3.1124
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    Abstract:

    In this investigation, the students’ goal was to isolate and characterize strains from soil. Following selection and enrichment on 1A-t medium, putative isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR) with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA). Primer sets included AGRH to identify isolates that were members of the , BIOVAR1 primers to identify members of biovar group I, and a third set, VIRG, to determine presence of (only present in pathogenic strains). During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of –plant interactions ( ≤ 0.001) and molecular biology ( ≤ 0.01). The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores ( ≤ 0.0001) and attainment of capstone experience learning outcomes.

Key Concept Ranking

Agrobacterium tumefaciens
0.4778626
Agarose Gel Electrophoresis
0.46784073
Molecular Techniques
0.45797458
Biochemical Test
0.44135296
0.4778626

References & Citations

1. Benzle KA, et al2015Isolation and characterization of novel Agrobacterium strains for soybean and sunflower transformationPlant Cell Tissue Organ Cult121718110.1007/s11240-014-0679-x http://dx.doi.org/10.1007/s11240-014-0679-x
2. Brisbane PG, Kerr A1983Selective media for three biovars of AgrobacteriumJ Appl Bacteriol5442543110.1111/j.1365-2672.1983.tb02638.x http://dx.doi.org/10.1111/j.1365-2672.1983.tb02638.x
3. Gelvin SB2003Agrobacterium-mediated plant transformation: the biology behind the gene-jockeying toolMicrobiol Mol Biol Rev67163710.1128/MMBR.67.1.16-37.2003150518 http://dx.doi.org/10.1128/MMBR.67.1.16-37.2003
4. Hood EE, Gelvin SB, Melchers LS, Hoekema A1993New Agrobacterium helper plasmids for gene transfer to plantsTrans Res220821810.1007/BF01977351 http://dx.doi.org/10.1007/BF01977351
5. Krathwohl DR2002A revision of Bloom’s taxonomy: an overviewTheory Pract4121221810.1207/s15430421tip4104_2 http://dx.doi.org/10.1207/s15430421tip4104_2
6. Lennox JR1980Agrobacterium and tumor induction: a model systemAm Biol Teach4216016510.2307/4446860 http://dx.doi.org/10.2307/4446860
7. Martinez-Vaz BM, Denny R, Young ND, Sadowsky MJ2015An alternative approach to “identification of unknowns”: designing a protocol to verify the identities of nitrogen fixing bacteriaJ Microbiol Biol Educ16224725310.1128/jmbe.v16i2.973 http://dx.doi.org/10.1128/jmbe.v16i2.973
8. Minnemeyer SL, Lightfoot R, Matthysse AG1991A semiquantitative bioassay for relative virulence of Agrobacterium tumefaciens strains on Bryophyllum daigremontianaJ Bacteriol1737723772410.1128/jb.173.23.7723-7724.19911938971212546 http://dx.doi.org/10.1128/jb.173.23.7723-7724.1991
9. Mougel C, Cournoyer B, Nesme X2001Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2Appl Environ Microbiol67657410.1128/AEM.67.1.65-74.20011113342992517 http://dx.doi.org/10.1128/AEM.67.1.65-74.2001
10. Palmer ACV, Shaw CH1992The role of VirA and VirG phosphorylation in chemotaxis towards acetosyringone by Agrobacterium tumefaciensJ Gen Microbiol1382509251410.1099/00221287-138-12-2509 http://dx.doi.org/10.1099/00221287-138-12-2509
11. Wilson DE, Chosewood LC2009Biosafety in microbiological and biomedical laboratories5th EditionCenter for Disease Control and Prevention publication number 21–1112Center for Disease Control and PreventionAtlanta, GA
12. Yanagi M, Yamasato K1993Phylogenic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencerFEMS Microbiol Lett10711512010.1111/j.1574-6968.1993.tb06014.x7682191 http://dx.doi.org/10.1111/j.1574-6968.1993.tb06014.x
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2016-12-02
2017-11-22

Abstract:

In this investigation, the students’ goal was to isolate and characterize strains from soil. Following selection and enrichment on 1A-t medium, putative isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR) with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA). Primer sets included AGRH to identify isolates that were members of the , BIOVAR1 primers to identify members of biovar group I, and a third set, VIRG, to determine presence of (only present in pathogenic strains). During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of –plant interactions ( ≤ 0.001) and molecular biology ( ≤ 0.01). The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores ( ≤ 0.0001) and attainment of capstone experience learning outcomes.

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Figures

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FIGURE 1

(A) Soil samples plated on 1A-t medium. Colonies are putative isolates. (B) Positive oxidase tests.

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 444-450. doi:10.1128/jmbe.v17i3.1124
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FIGURE 2

Test results for 3-ketolactose. (A) Positive control, EHA105. (B) 1, 2: negative isolates; 3: positive isolate

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 444-450. doi:10.1128/jmbe.v17i3.1124
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Image of FIGURE 3

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FIGURE 3

Gel electrophoresis of PCR products. Lane 6 = 100 bp ladder. Lanes 3, 4, 5, 8, 9 primer controls (3 AGRH; 4, 8 BIOVAR1; 5, 9 VIRG). Student amplification products are in lanes 2 (BIOVAR1), 7 (AGRH), and 11 (BIOVAR1). The student sample in lane 10 contains amplicons from all three primer sets. Lanes 1 and 12 are empty. PCR = polymerase chain reaction.

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 444-450. doi:10.1128/jmbe.v17i3.1124
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Image of FIGURE 4

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FIGURE 4

-induced gall (arrow) formed in .

Source: J. Microbiol. Biol. Educ. December 2016 vol. 17 no. 3 444-450. doi:10.1128/jmbe.v17i3.1124
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