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

    Authors: Kathleen Sandman1,*, Christopher Ecker1
    VIEW AFFILIATIONS HIDE AFFILIATIONS
    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: sandman.1@osu.edu.
    • ©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 PAHM2012The rhizosphere microbiome and plant healthTrends Plant Sci17847848610.1016/j.tplants.2012.04.00122564542 http://dx.doi.org/10.1016/j.tplants.2012.04.001
2. Burr SE, Gobeli S, Kuhnert P, Goldschmidt-Clermont E, Frey J2010Pseudomonas chlororaphis subsp. piscium subsp. nov., isolated from freshwater fishInt. J. System. Evol. Microbiol60122753275710.1099/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 BJ2007Pseudomonas reinekei sp. nov., Pseudomonas moorei sp. nov. and Pseudomonas mohnii sp. nov., novel species capable of degrading chlorosalicylates or isopimaric acidInt. J. System. Evol. Microbiol57592393110.1099/ijs.0.64703-0 http://dx.doi.org/10.1099/ijs.0.64703-0
4. Cole JR, et al2013Ribosomal Database Project: data and tools for high throughput rRNA analysisNucleic Acids Res42D1D633D64210.1093/nar/gkt1244242883683965039 http://dx.doi.org/10.1093/nar/gkt1244
5. Gormally C, Brickman P, Hallar B, Armstrong N2009Effects of inquiry-based learning on students’ science literacy skills and confidenceInt. J. Scholarship Teach. Learn3216
6. Höfte M, deVos PPlant pathogenic Pseudomonas species2006 Gnanamanickam SSPlant-Associated BacteriaSpringer10.1007/978-1-4020-4538-7_14 http://dx.doi.org/10.1007/978-1-4020-4538-7_14
7. Liao CS, Chen LC, Chen BS, Lin SH2010Bioremediation of endocrine disruptor di-n-butyl phthalate ester by Deinococcus radiodurans and Pseudomonas stutzeriChemosphere78334234610.1016/j.chemosphere.2009.10.020 http://dx.doi.org/10.1016/j.chemosphere.2009.10.020
8. McDonald D, et al2012An improved GreenGenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaeaISME J661061810.1038/ismej.2011.1393280142 http://dx.doi.org/10.1038/ismej.2011.139
9. Mulet M, Lalucat J, García-Valdés E2010DNA sequence-based analysis of the Pseudomonas speciesEnviron. Microbiol1261513153020192968
10. Mulet M, David Z, Nogales B, Bosch R, Lalucat J, García-Valdés E2011Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spillAppl. Environ. Microbiol7731076108510.1128/AEM.01741-103028729 http://dx.doi.org/10.1128/AEM.01741-10
11. Quast C, et al2013The SILVA ribosomal RNA gene database project: improved data processing and web-based toolsNucleic Acids Res41D1D590D59610.1093/nar/gks12193531112 http://dx.doi.org/10.1093/nar/gks1219
12. Santoyo G, Orozco-Mosqueda Md, Govindappa M2012Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: a reviewBiocontrol Sci. Technol22885587210.1080/09583157.2012.694413 http://dx.doi.org/10.1080/09583157.2012.694413
13. Sharma S, Pathak J2014Pseudomonas in biodegradationInt. J. Pure Appl. Biosci22213222
14. Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kämpfer P2010Notes on the characterization of prokaryote strains for taxonomic purposesInt. J. System. Evol. Microbiol60124926610.1099/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 J1996Polyphasic taxonomy, a consensus approach to bacterial systematicsMicrobiol. Mol. Biol. Rev602407438
16. Widmer F, Seidler RJ, Gillevet PM, Watrud LS, DiGiovanni GD1998A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (Sensu Stricto) in environmental samplesAppl. Environ. Microbiol647254525539647828106424
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/content/journal/jmbe/10.1128/jmbe.v15i2.754
2014-12-15
2017-07-27

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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