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An Undergraduate Laboratory Activity Demonstrating Bacteriophage Specificity

    Authors: Mary E. Allen1, Ruth A. Gyure2,*
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    Affiliations: 1: Department of Biology, Hartwick College, Oneonta, NY 13820; 2: Department of Biological and Environmental Sciences, Western Connecticut State University, Danbury CT 06810
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 06 May 2013
    • Supplemental materials available at http://jmbe.asm.org
      Primary incoming sewage is untreated and can be obtained from a local sewage treatment plant upon request. The treatment plant operator/director is typically quite cooperative and should of course be contacted beforehand to obtain permission and to explain the educational purpose for collecting the sample.
    • *Corresponding author. Mailing address: Department of Biological and Environmental Sciences, Western Connecticut State University, Danbury CT 06810. Phone: 203-837-8796. Fax: 203-837-8769. E-mail: gyurer@wcsu.edu.
    • ©2013 Author(s). Published by the American Society for Microbiology.
    Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 84-92. doi:10.1128/jmbe.v14i1.534
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    Abstract:

    Bacteriophage are among the most diverse and numerous microbes inhabiting our planet. Yet many laboratory activities fail to engage students in meaningful exploration of their diversity, unique characteristics, and abundance. In this curriculum activity students use a standard plaque assay to enumerate bacteriophage particles from a natural sample and use the scientific method to address questions about host specificity and diversity. A raw primary sewage sample is enriched for bacteriophage using hosts in the family Enterobacteriaceae. Students hypothesize about host specificity and use quantitative data (serial dilution and plaque assay) to test their hypotheses. Combined class data also help them answer questions about phage diversity. The exercise was field tested with a class of 47 students using pre- and posttests. For all learning outcomes posttest scores were higher than pretest scores at or below = 0.01. Average individualized learning gain (G) was also calculated for each learning outcome. Students’ use of scientific language in reference to bacteriophage and host interaction significantly improved ( = 0.002; G = 0.50). Improved means of expression helped students construct better hypotheses on phage host specificity (G = 0.31, = 0.01) and to explain the plaque assay method (G = 0.33, = 0.002). At the end of the exercise students also demonstrated improved knowledge and understanding of phage specificity as related to phage therapy in humans ( < 0.001; G = 51).

Key Concept Ranking

Wastewater Treatment Plants
0.42902377
Escherichia coli
0.42857137
Serratia marcescens
0.4084249
0.42902377

References & Citations

1. d’Herelle F1917Sur un microbe invisible anatagoniste des bacilles dysenteriqueAcad Sci Paris165373375
2. Dulbecco R, Vogt M1953Some problems of animal virology as studied by the plaque techniqueCold Spring Harb Symp Quant Biol1827327910.1101/SQB.1953.018.01.03913168995 http://dx.doi.org/10.1101/SQB.1953.018.01.039
3. Emmert EABthe ASM Task Committee on Laboratory Biosafety2013Biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationaleJ. Microbiol. Biol. Educ147883
4. Gratia A1936Numerical relations between lysogenic bacteria and particles of bacteriophageAnn Inst Pasteur57652
5. Hershey AD, Kalmanson G, Bronfenbrenner J1943Quantitative methods in the study of the phageantiphage reactionJ Immunol46267279
6. Katz DS, Panec M2006Plaque assay: assorted views of bacteriophage platesAmerican Society for Microbiologyhttp://www.microbelibrary.org. Accessed 22 September 2012.
7. Panec M, Katz DS2006Plaque assay protocolsAmerican Society for Microbiologyhttp://www.microbelibrary.org. Accessed 21 September 2012.
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/content/journal/jmbe/10.1128/jmbe.v14i1.534
2013-05-06
2017-05-27

Abstract:

Bacteriophage are among the most diverse and numerous microbes inhabiting our planet. Yet many laboratory activities fail to engage students in meaningful exploration of their diversity, unique characteristics, and abundance. In this curriculum activity students use a standard plaque assay to enumerate bacteriophage particles from a natural sample and use the scientific method to address questions about host specificity and diversity. A raw primary sewage sample is enriched for bacteriophage using hosts in the family Enterobacteriaceae. Students hypothesize about host specificity and use quantitative data (serial dilution and plaque assay) to test their hypotheses. Combined class data also help them answer questions about phage diversity. The exercise was field tested with a class of 47 students using pre- and posttests. For all learning outcomes posttest scores were higher than pretest scores at or below = 0.01. Average individualized learning gain (G) was also calculated for each learning outcome. Students’ use of scientific language in reference to bacteriophage and host interaction significantly improved ( = 0.002; G = 0.50). Improved means of expression helped students construct better hypotheses on phage host specificity (G = 0.31, = 0.01) and to explain the plaque assay method (G = 0.33, = 0.002). At the end of the exercise students also demonstrated improved knowledge and understanding of phage specificity as related to phage therapy in humans ( < 0.001; G = 51).

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Figures

Image of FIGURE 1

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FIGURE 1

Primary sewage sample. This photograph shows an example of a sampling container. In this case it is a clean (bleached) one-pint mason jar properly labeled with a tight fitting lid. The sample is collected then placed in a secondary container for transport back to the laboratory.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 84-92. doi:10.1128/jmbe.v14i1.534
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Image of FIGURE 2

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FIGURE 2

Filtration step. This photograph shows a sterile disposable 0.45-μm filter tower (150-ml size) which makes filtration of the sewage enrichments convenient. The filtrate collected constitutes the phage lysate that will be distributed for class use.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 84-92. doi:10.1128/jmbe.v14i1.534
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Image of FIGURE 3

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FIGURE 3

Tubes prepared for students. These disposable 15-ml tubes are clearly labeled as CULTURE (or HOST) and LYSATE (or PHAGE) to avoid any confusion during the activity.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 84-92. doi:10.1128/jmbe.v14i1.534
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Image of FIGURE 4

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FIGURE 4

The student station. Each station should be equipped with the following: 9 ml saline; three empty 15-ml tubes with screw caps; three disposable sterile 1-ml pipettes and pipetting device; five or six disposable sterile transfer pipettes; three TS agar plates; gloves, goggles, and biowaste container. Also note the freshly prepared CULTURE and LYSATE.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 84-92. doi:10.1128/jmbe.v14i1.534
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Image of FIGURE 5

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FIGURE 5

Rubric scores for six targeted learning outcomes. Averages (n = 47) of student scores on a rubric ( Appendix 4 ) used to measure knowledge of the six learning outcomes on a scale of 1–4. Learning was measured with pre- and posttests. Learning outcome 2 is divided into three parts: LOH = hypothesis, LOM = method, LOA = analysis. Two-tailed t-tests indicated differences between all pre- and posttest averages were significant at, or below, = 0.01.

Source: J. Microbiol. Biol. Educ. May 2013 vol. 14 no. 1 84-92. doi:10.1128/jmbe.v14i1.534
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