1887

Pseudomonas Isolation and Identification: An Introduction to the Challenges of Polyphasic Taxonomy

    Authors: Kathleen Sandman1,*, Christopher Ecker1
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    Affiliations: 1: Department of Microbiology, The Ohio State University, Columbus, OH 43210-1292
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 15 December 2014
    • Supplemental materials available at http://jmbe.asm.org
    • *Corresponding author. Mailing address: Department of Microbiology, The Ohio State University, 484 W. 12th Avenue, Room 105, Columbus, OH 43210-1292. Phone: 614-292-5867. Fax: 614-292-8120. E-mail: [email protected].
    • ©2014 Author(s). Published by the American Society for Microbiology.
    Source: J. Microbiol. Biol. Educ. December 2014 vol. 15 no. 2 287-291. doi:10.1128/jmbe.v15i2.754
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    Abstract:

    The ability to isolate an organism in pure culture from the environment is a manageable task for undergraduate students; the identification of that organism requires integration of both genotypic and phenotypic data and illustrates the challenges inherent in contemporary bacterial taxonomy. In this ten-laboratory period series of exercises, students isolate a strain of from soil and characterize its biochemical and physiological properties, as well as determine the DNA sequence of its 16S rRNA genes. Integrating these data positions students to defend their classification of the isolate as a new species or as a member of a validly described species. Assessment data demonstrate that both knowledge of and confidence in understanding of the principles of laboratory handling of and bacterial taxonomy increased following the exercises.

Key Concept Ranking

16s rRNA Sequencing
0.53141546
Agarose Gel Electrophoresis
0.4854109
Bacterial Classification
0.4834543
Chromosomal DNA
0.46301553
0.53141546

References & Citations

1. Berendsen RL, Pieterse CMJ, Bakker PAHM 2012 The rhizosphere microbiome and plant health Trends Plant Sci 17 8 478 486 10.1016/j.tplants.2012.04.001 22564542 http://dx.doi.org/10.1016/j.tplants.2012.04.001
2. Burr SE, Gobeli S, Kuhnert P, Goldschmidt-Clermont E, Frey J 2010 Pseudomonas chlororaphis subsp. piscium subsp. nov., isolated from freshwater fish Int. J. System. Evol. Microbiol 60 12 2753 2757 10.1099/ijs.0.011692-0 http://dx.doi.org/10.1099/ijs.0.011692-0
3. Camara B, Strompl C, Verbarg S, Sproer C, Pieper DH, Tindall BJ 2007 Pseudomonas reinekei sp. nov., Pseudomonas moorei sp. nov. and Pseudomonas mohnii sp. nov., novel species capable of degrading chlorosalicylates or isopimaric acid Int. J. System. Evol. Microbiol 57 5 923 931 10.1099/ijs.0.64703-0 http://dx.doi.org/10.1099/ijs.0.64703-0
4. Cole JR, et al 2013 Ribosomal Database Project: data and tools for high throughput rRNA analysis Nucleic Acids Res 42 D1 D633 D642 10.1093/nar/gkt1244 24288368 3965039 http://dx.doi.org/10.1093/nar/gkt1244
5. Gormally C, Brickman P, Hallar B, Armstrong N 2009 Effects of inquiry-based learning on students’ science literacy skills and confidence Int. J. Scholarship Teach. Learn 3 2 16
6. Höfte M, deVos P Plant pathogenic Pseudomonas species 2006 Gnanamanickam SS Plant-Associated Bacteria Springer 10.1007/978-1-4020-4538-7_14 http://dx.doi.org/10.1007/978-1-4020-4538-7_14
7. Liao CS, Chen LC, Chen BS, Lin SH 2010 Bioremediation of endocrine disruptor di-n-butyl phthalate ester by Deinococcus radiodurans and Pseudomonas stutzeri Chemosphere 78 3 342 346 10.1016/j.chemosphere.2009.10.020 http://dx.doi.org/10.1016/j.chemosphere.2009.10.020
8. McDonald D, et al 2012 An improved GreenGenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea ISME J 6 610 618 10.1038/ismej.2011.139 3280142 http://dx.doi.org/10.1038/ismej.2011.139
9. Mulet M, Lalucat J, García-Valdés E 2010 DNA sequence-based analysis of the Pseudomonas species Environ. Microbiol 12 6 1513 1530 20192968
10. Mulet M, David Z, Nogales B, Bosch R, Lalucat J, García-Valdés E 2011 Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill Appl. Environ. Microbiol 77 3 1076 1085 10.1128/AEM.01741-10 3028729 http://dx.doi.org/10.1128/AEM.01741-10
11. Quast C, et al 2013 The SILVA ribosomal RNA gene database project: improved data processing and web-based tools Nucleic Acids Res 41 D1 D590 D596 10.1093/nar/gks1219 3531112 http://dx.doi.org/10.1093/nar/gks1219
12. Santoyo G, Orozco-Mosqueda Md, Govindappa M 2012 Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: a review Biocontrol Sci. Technol 22 8 855 872 10.1080/09583157.2012.694413 http://dx.doi.org/10.1080/09583157.2012.694413
13. Sharma S, Pathak J 2014 Pseudomonas in biodegradation Int. J. Pure Appl. Biosci 2 2 213 222
14. Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P 2010 Notes on the characterization of prokaryote strains for taxonomic purposes Int. J. System. Evol. Microbiol 60 1 249 266 10.1099/ijs.0.016949-0 http://dx.doi.org/10.1099/ijs.0.016949-0
15. Vandamme P, Pot B, Gillis M, deVos P, Kersters K, Swings J 1996 Polyphasic taxonomy, a consensus approach to bacterial systematics Microbiol. Mol. Biol. Rev 60 2 407 438
16. Widmer F, Seidler RJ, Gillevet PM, Watrud LS, DiGiovanni GD 1998 A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (Sensu Stricto) in environmental samples Appl. Environ. Microbiol 64 7 2545 2553 9647828 106424

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2014-12-15
2019-04-25

Abstract:

The ability to isolate an organism in pure culture from the environment is a manageable task for undergraduate students; the identification of that organism requires integration of both genotypic and phenotypic data and illustrates the challenges inherent in contemporary bacterial taxonomy. In this ten-laboratory period series of exercises, students isolate a strain of from soil and characterize its biochemical and physiological properties, as well as determine the DNA sequence of its 16S rRNA genes. Integrating these data positions students to defend their classification of the isolate as a new species or as a member of a validly described species. Assessment data demonstrate that both knowledge of and confidence in understanding of the principles of laboratory handling of and bacterial taxonomy increased following the exercises.

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Figures

Image of FIGURE 1.

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FIGURE 1.

A. Streak from the liquid enrichment on a solid medium of the same composition, as observed following Lab 2. These are not pure cultures and a variety of colony morphologies are visible. B and C. Student streaks on Isolation Agar as observed following Lab 3. B is not yet a pure culture; C is a pure culture. D. Agarose gel image of genomic DNA preparations, annotated to show the molecular weight ladder (2 log ladder with bright bands at 3.0, 1.0, 0.5 kb), chromosomal DNA fragments (C) and plasmid band (P).

Source: J. Microbiol. Biol. Educ. December 2014 vol. 15 no. 2 287-291. doi:10.1128/jmbe.v15i2.754
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Image of FIGURE 2.

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FIGURE 2.

The percentage of students answering correctly in the pretest (green) vs. the posttest (blue). For each bar the dark lower portion illustrates the percentage of students who were both correct and confident; the lighter upper portion denotes the percentage who were correct but guessing. The questions are included in Appendix 6 .

Source: J. Microbiol. Biol. Educ. December 2014 vol. 15 no. 2 287-291. doi:10.1128/jmbe.v15i2.754
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