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Introduction.
Topics related to the ecology of microorganisms are of increasing interest in the undergraduate curriculum. This module, a set of three exercises, is designed to provide a resource for a lab or classroom activity that addresses microbial ecology at the level of sampling strategy and experimental design. These exercises will be relevant in courses where microbial ecology is addressed (e.g., general biology, microbiology, ecology) or where microbial ecology examples can be used to teach experimental design related to ecology. A wide variety of accessible ecosystems may be utilized if the instructor wishes to move on to apply the design in a laboratory or field exercise.
This activity may also be helpful for instructors in preparing students for doing independent research in other areas of biology. By doing these exercises, students will gain an appreciation for many fundamental principles of sound experimental design, measurement, and analysis and will avoid some common pitfalls in the planning and execution of more serious (and costly) projects.
Learning Objectives.
1. Identify challenges that microbial ecologists face in measuring and sampling for microbial presence and activity in the natural environment (Introduction)
2. Differentiate between species diversity and community structure as applied to microbial ecology (Activity 1)
3. Learn a method for quantifying diversity in a microbial community (Activity 1)
4. Design an appropriate sampling scheme that takes into account community structure and organization (temporal and spatial) as well as scaling issues (Activity 2)
5. Recognize how to build replication into experimental design and avoid some common pitfalls when sampling and measuring microbial systems (Activity 3)
Background.
The background knowledge students should have to complete this activity is embedded within it and additional preparation of the students is not necessary. Some major considerations when studying microorganisms in the environment are: how do we detect them, how do we identify them, and how do we quantify their presence. The following considerations are addressed in the three exercises that comprise this activity.
1. Detection and visualization of microscopic organisms. Because direct visualization of microbes in a representational manner is difficult, this challenge emphasizes the importance of a very sound sampling strategy when obtaining microbes from the natural environment and making claims about their roles and numbers.
2. Definition and differentiation of microbial taxa. Microbes are not easily classified into species or even genera. Molecular methods of identification are helpful but can also introduce a different sort of sampling and analytical bias.
3. Cultivation issues and ecological relevance. Are the microbes captured and cultivated truly those that are most important in the ecosystem? How can we cultivate and study organisms whose needs we cannot identify?
4. Interdependence. Microbial interactions are often complex and can be extremely difficult to sort out. For example, consortia are pairs or groups of species interacting in such a tightly coupled manner that separation is nearly impossible due to metabolic interdependence.
5. Adherence to surfaces. Microbes most typically grow on surfaces—attached to mucosa, rock, plastic, wood, dust and soil particles, etc. Even in open water, the majority of microbes are surface attached on microscopic particles of organic or inorganic matter that provide energy and other nutritional compounds. This adherent state must be taken into account when designing sampling and measurement strategies.
The exercises are organized to address these issues in the following manner:
Exercise 1. Measuring diversity
When sampling an unknown microbial community, the researcher first must determine the level of variation and heterogeneity in the target area. Some initial sampling and analysis will help in answering the question: how many samples must be taken in order to best describe the diversity of the community? In this exercise, students do simulated sampling with simple materials in the classroom and construct a graph that quantitatively helps them make an informed decision about the community and sampling effort required. They quantify the diversity of their communities using the Simpson's index of diversity, a measure commonly used in ecology.
Exercise 2. Community structure and organization
In the heterogeneous microbial world, cells organize and interact at both small scale and higher levels based upon factors that influence them and the resources available. In this exercise, which compliments and extends Exercise 1, students will do a simulated sampling and analysis that emphasizes the spatial organization of microbes in the environment and how these considerations inform sampling approach.
Exercise 3. Common pitfalls in the study of microbial communities—pseudoreplication
After making a decision about how to sample and how many samples are needed, a next step is to make sure that experiments are replicated. A major pitfall in the design of experiments is to fall into the trap of doing an “easy” replication, which may not be a true independent replicate at all. In this exercise, students are challenged with several experimental questions and design setups and learn to distinguish true independent replicates from pseudoreplicates that don’t contribute to the validity of experimental results.
PROCEDURE
Materials.
If each group does not have access to a computer, a calculator is desirable for computing Simpson’s index of diversity, if this will be done in class. Students will also need scrap paper and/or paper to record notes. For each exercise, a handout, diagrams, and instructor guide are provided.
Exercise 1 (per group of two to four students):
· A simulated “bag community” consisting of a lunch-size paper bag filled with different combinations of common objects such as beads, erasers, dry beans, etc.
· Graph paper
· Computers (allow for use of Excel spreadsheets) or calculators
Exercise 2 (per group of two to four students):
· Printout of sample community diagram A, B, C, or D (Word versions are included with the exercise)
· 1 Sharpie or other permanent marker
· 3 plastic transparencies, same size as the community diagram (8.5 x 11”)
· Data sheet (printed out, or electronically accessed on computer)
Exercise 3:
This is a thought activity. No special materials are required. A supplementary optional Powerpoint is included if the instructor wishes to project the diagrams during the activity.
Student Version.
Instructor Version.
Safety Issues.
None
Suggestions for Determining Student Learning.
I. Measurement of student learning in the classroom
These exercises were field tested in an undergraduate microbial ecology class. This was an upper-level biology course with 14 enrolled junior and senior biology and biochemistry majors. Prior to taking the course, all students had been exposed to a little ecology in general biology and some had more exposure in a previous ecology course. The exercises were done within the first month of the semester. Pre- and posttests were administered to students to measure what they had learned from the exercises.
At the end of the exercises an additional 20% of the students correctly identified the challenge of measuring and sampling for microbial presence and activity in the natural environment (65% of the total students). Identical results were obtained when students were asked to discriminate between measures of diversity (richness and evenness). Students were also 40% better at differentiating between species diversity and community structure. On the posttest all of the students reported strong agreement or agreement, that the activities helped them to understand difficulties in sampling microbial communities and differences between community structure and diversity.
When asked questions about experimental design 86 to 93% (it varied by question) of the students answered correctly on the posttest, compared with 28 to 50% on the pretest. Finally, before the exercises only one student was familiar with pseudoreplication and on the posttest 78% correctly defined this term. All but one of the students strongly agreed or agreed, that the activities helped them to understand how microbial diversity is measured and what it is that microbial ecologists do.
II. Assessment of usefulness of the activity (educators' evaluation)
These exercises were also done at the American Society for Microbiology Conference for Undergraduate Educators (ASMCUE) in May 2008. The audience members were heterogeneous in background, training, and experience related to microbial ecology. After completing the three exercises, participants were given a survey to assess their opinions about the value of the activity for classroom use and how well it addressed the learning objectives.
The data clearly show that this activity is highly suitable for undergraduates in both microbiology and general biology courses and will also be helpful in teaching general ecology and statistics (Fig. 1). Respondents overwhelmingly agreed that this activity will help students appreciate considerations involved in the experimental design process and will reinforce important quantitative issues in biology. It is highly suitable for large lecture classrooms as well as the laboratory and can be combined with a wet lab or field experience (Fig. 2). Out of 48 sampled, 42 participants agreed that the use of primary literature in the activity was helpful; six said it was “somewhat helpful."
Overall, this activity was seen by microbiology educators as a very useful one for teaching the targeted principles.
FIG. 1. Suitability of activity for different student audiences, based on a survey of participants at ASMCUE 2008; n = 47.
FIG. 2. Number of ASMCUE 2008 participants, n = 48, who agreed with the following statements:
(A) Will help students appreciate considerations of experimental design, (B) suitable for large lecture classroom, >40 students, (C) suitable for a laboratory exercise, (D) reinforces important quantitative issues in biology, (E) easily combined with a wet lab or field activity.
References.
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3. Desmarais, T. R., H. M. Solo-Gabriele, and C. J. Palmer. 2002. Influence of soil on fecal indicator organisms in a tidally influenced subtropical environment. Appl. Env. Microbiol. 68:1165–1172.
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7. Heffner, R. A., M. J. Butler IV, and C. Keelan Reilly. 1996. Pseudoreplication revisited. Ecology 77(8):2558–2562.
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12. Ranjard, L., D. P. H. Lejon, C. Mougel, L. Schehrer, D. Merdinoglu, and R. Chaussod. 2003. Sampling strategy in molecular microbial ecology: influence of soil sample size on DNA fingerprinting analysis of fungal and bacterial communities. Environ. Microbiol. 5:1111–1120.
13. Schloss, P. D., and J. Handelsman. 2007. The last word: books as a statistical metaphor for microbial communities. Annu. Rev. Microbiol. 61:23–34.
14. Schloss, P. D., B. R. Larget, and J. Handelsman. 2004. Integration of microbial ecology and statistics: a test to compare gene libraries. Appl. Environ. Microbiol. 70:5485–5492.
15. Torsvik, V., L. Ovreas, and T. F. Thingstad. 2002. Prokaryotic diversity—magnitude, dynamics, and controlling factors. Science 296:1064–1066.
16. Whitfield, J. 2005. Biogeography: is everything everywhere? News Briefs in Science 310:960–961. (Reports on the work of Bland Findlay, Centre for Ecology and Hydrology, Dorset, UK)
17. Yamamoto, M., H. Murai, A. Takeda, S. Okunishi, and H. Morisaki. 2005. Bacterial flora of the biofilm formed on the submerged surface of the reed Phragmites australis. Microbes Environ. 20(1):14–24.
Appendices.
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