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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: [email protected].
    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 al 2015 Isolation and characterization of novel Agrobacterium strains for soybean and sunflower transformation Plant Cell Tissue Organ Cult 121 71 81 10.1007/s11240-014-0679-x http://dx.doi.org/10.1007/s11240-014-0679-x
2. Brisbane PG, Kerr A 1983 Selective media for three biovars of Agrobacterium J Appl Bacteriol 54 425 431 10.1111/j.1365-2672.1983.tb02638.x http://dx.doi.org/10.1111/j.1365-2672.1983.tb02638.x
3. Gelvin SB 2003 Agrobacterium-mediated plant transformation: the biology behind the gene-jockeying tool Microbiol Mol Biol Rev 67 16 37 10.1128/MMBR.67.1.16-37.2003 150518 http://dx.doi.org/10.1128/MMBR.67.1.16-37.2003
4. Hood EE, Gelvin SB, Melchers LS, Hoekema A 1993 New Agrobacterium helper plasmids for gene transfer to plants Trans Res 2 208 218 10.1007/BF01977351 http://dx.doi.org/10.1007/BF01977351
5. Krathwohl DR 2002 A revision of Bloom’s taxonomy: an overview Theory Pract 41 212 218 10.1207/s15430421tip4104_2 http://dx.doi.org/10.1207/s15430421tip4104_2
6. Lennox JR 1980 Agrobacterium and tumor induction: a model system Am Biol Teach 42 160 165 10.2307/4446860 http://dx.doi.org/10.2307/4446860
7. Martinez-Vaz BM, Denny R, Young ND, Sadowsky MJ 2015 An alternative approach to “identification of unknowns”: designing a protocol to verify the identities of nitrogen fixing bacteria J Microbiol Biol Educ 16 2 247 253 10.1128/jmbe.v16i2.973 http://dx.doi.org/10.1128/jmbe.v16i2.973
8. Minnemeyer SL, Lightfoot R, Matthysse AG 1991 A semiquantitative bioassay for relative virulence of Agrobacterium tumefaciens strains on Bryophyllum daigremontiana J Bacteriol 173 7723 7724 10.1128/jb.173.23.7723-7724.1991 1938971 212546 http://dx.doi.org/10.1128/jb.173.23.7723-7724.1991
9. Mougel C, Cournoyer B, Nesme X 2001 Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2 Appl Environ Microbiol 67 65 74 10.1128/AEM.67.1.65-74.2001 11133429 92517 http://dx.doi.org/10.1128/AEM.67.1.65-74.2001
10. Palmer ACV, Shaw CH 1992 The role of VirA and VirG phosphorylation in chemotaxis towards acetosyringone by Agrobacterium tumefaciens J Gen Microbiol 138 2509 2514 10.1099/00221287-138-12-2509 http://dx.doi.org/10.1099/00221287-138-12-2509
11. Wilson DE, Chosewood LC 2009 Biosafety in microbiological and biomedical laboratories 5th Edition Center for Disease Control and Prevention publication number 21–1112 Center for Disease Control and Prevention Atlanta, GA
12. Yanagi M, Yamasato K 1993 Phylogenic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer FEMS Microbiol Lett 107 115 120 10.1111/j.1574-6968.1993.tb06014.x 7682191 http://dx.doi.org/10.1111/j.1574-6968.1993.tb06014.x

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2016-12-02
2019-07-24

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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Image of FIGURE 2

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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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