An undergraduate laboratory activity demonstrating bacteriophage specificity

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 p = 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 (p = 0.002; G = 0.50). Improved means of expression helped students construct better hypotheses on phage host specificity (G = 0.31, p = 0.01) and to explain the plaque assay method (G = 0.33, p = 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 (p < 0.001; G = 51).

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.

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.

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.

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.

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, p = 0.01.