JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION, December 2015, p. 211-216 DOI: http://dx.doi.org/10.1128/jmbe.v16i2.926
Curriculum
Discovery of the Collaborative Nature of Science with Undergraduate Science Majors and Non-Science Majors through the Identification of Microorganisms Enriched in Winogradsky Columns † Jasmine Ramirez1, Catalina Arango Pinedo1, and Brian M. Forster2* of Biology, Saint Joseph’s University, Philadelphia, PA 19131, 2College of Arts & Sciences, Saint Joseph’s University, Philadelphia, PA 19131 1Department
Today’s science classrooms are addressing the need for non-scientists to become scientifically literate. A key aspect includes the recognition of science as a process for discovery. This process relies upon interdisciplinary collaboration. We designed a semester-long collaborative exercise that allows science majors taking a general microbiology course and non-science majors taking an introductory environmental science course to experience collaboration in science by combining their differing skill sets to identify microorganisms enriched in Winogradsky columns. These columns are self-sufficient ecosystems that allow researchers to study bacterial populations under specified environmental conditions. Non-science majors identified phototrophic bacteria enriched in the column by analyzing the signature chlorophyll absorption spectra whereas science majors used 16S rRNA gene sequencing to identify the general bacterial diversity. Students then compiled their results and worked together to generate lab reports with their final conclusions identifying the microorganisms present in their column. Surveys and lab reports were utilized to evaluate the learning objectives of this activity. In pre-surveys, nonmajors’ and majors’ answers diverged considerably, with majors providing responses that were more accurate and more in line with the working definition of collaboration. In post-surveys, the answers between majors and nonmajors converged, with both groups providing accurate responses. Lab reports showed that students were able to successfully identify bacteria present in the columns. These results demonstrate that laboratory exercises designed to group students across disciplinary lines can be an important tool in promoting science education across disciplines.
INTRODUCTION With the ongoing discussion of American scientific illiteracy in mind, we have designed a semester-long experiment that promotes the collaborative nature of science as a means of enhancing science education. Students have the misconception that today’s scientists are highly specialized workers in a specific area. Rather, scientists collaborate by exchanging ideas and seek the opinions of others outside their field to answer a question. Recommendations on learning and teaching suggest that teachers should promote collaboration through group work, and that there should be an emphasis on the idea that science is a collection of different scientific fields or content disciplines (8). Therefore, it is important to have students participate in multi-field experiments. Incorporating collaboration in the classroom can benefit students, especially in classes that have both science *Corresponding author. Mailing address: 5600 City Avenue, 112 Connelly Hall, Philadelphia, PA 19131. Phone: 610-660-3188. E-mail: [email protected]. †Supplemental materials available at http://jmbe.asm.org
and non-science major students (4, 9, 10). The National Research Council has reported that STEM students quickly specialize in one specific discipline while other students take a year or less of STEM courses (7). To address this, science curricula should seek to foster collaborative learning so that both STEM and non-STEM students can have their learning experience enhanced and understand the benefits of collaboration in the scientific process. This paper describes a semester-long laboratory exercise where science majors and non-science majors collaborate to construct and analyze Winogradsky columns. This ecosystem provides a convenient means for studying the growth requirements of bacteria (2). Columns are packed with soil and water. The soil can be amended with cellulose and additional carbon (organic and/or inorganic) and sulfur sources to enrich for various types of autotrophic and heterotrophic bacteria. In the column, the organic material at the bottom of the column is used up, resulting in depleted O2 at the bottom of the column and higher levels of O2 at the top. The anaerobes that are now selected for at the bottom of the column produce carbon by-products which are then used by sulfate-utilizing bacteria to produce H2S.
©2015 Author(s). Published by the American Society for Microbiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
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This results in high concentrations of H2S at the bottom of the column and lower levels at the top. The H2S is then available for cellular respiration or as an electron source for photosynthesis. The column promotes the growth of different bacterial types at various depths corresponding to O2 and H2S concentrations. The columns are subsequently incubated in front of a light source to enrich for phototrophic bacteria for approximately six to eight weeks. This exercise teaches students two different methods of identifying bacterial species in the column. One method is extracting chlorophyll and analyzing its absorbance spectra. This is a very broad method for identification and is restricted to the identification of phototrophs, both bacteria and eukaryotes. The second method is sequencing the gene that encodes the 16S ribosomal ribonucleic acid (rRNA) found in the small ribosomal unit of bacteria and identifying a spectrum of bacterial species using basic local alignment search tool (BLAST) analysis (1). A previous exercise has been published which describes the use of the chlorophyll identification as a means of identifying enriched anoxic phototrophic microorganisms from environmental samples (3). In that study, pure cultures of oxygenic phototrophic bacteria were utilized. Following the chlorophyll identification of the samples, students used microscopy to visualize microorganisms from their enrichments. In this lab exercise, we allow students to enrich for both oxic and anoxic microorganisms and have groups supplement their chlorophyll identification with 16S rRNA gene analysis.
instructors should plan to have their students work on other laboratory activities that may or may not be related to this activity. Groups should independently make their observations once a week. Observations are expected to include writing and drawings depicting changes in soil color. During the incubation period between weeks 2 through 7, science majors should learn about deoxyribonucleic acid (DNA) isolation, polymerase chain reaction (PCR), and 16S rRNA gene sequencing. Non-science majors should learn about chlorophyll and phototrophic bacteria. In the eighth week, groups meet to discuss what they have learned and open their columns. Non-science majors identify phototrophic microorganisms through analysis of the chlorophyll absorbance spectra. This procedure will take approximately one hour. Science majors isolate genomic DNA from the soil and identify bacteria by 16S rRNA gene sequencing. This will take three lab sessions of three hours each. During their last meeting of the semester, group members discuss their results and prepare lab reports (Appendices 1 and 2). While majors and nonmajors collaborate on their data and discuss with one another the strengths and weaknesses of each technique used, they each prepare separate reports and are graded on expectations commensurate with their level of scientific knowledge.
Intended audience/Prerequisite student knowledge
1. D efine the term collaboration as it applies to the nature of science. 2. Name two different techniques for identifying microorganisms. 3. Use absorbance spectra of (bacterio)chlorophyll and 16S rRNA gene sequencing to identify microorganisms that have been enriched in a Winogradsky column.
This activity was designed for groups consisting of biology majors (general microbiology students) and non-science majors (introductory environmental science students). Both groups of students should have a basic understanding of the fact that bacteria can be found in different environments depending upon their nutritional and gas requirements in order to grow and of the process of photosynthesis. They should also be familiar with aseptic technique. While there is no additional prerequisite knowledge for non-science majors, science majors should be familiar with what it means for a bacterium to be defined as a phototroph and with basic concepts of genetics. This type of exercise may be appropriate for science lab-based courses of any level, including microbiology classes. This activity can also be used in large class settings, with modifications described in Appendix 2. Learning time This activity has seven parts spanning an entire academic semester. Science major and non-science major students meet three times in the semester. Labs meet for three hours, once a week. In the first week, the students (a) discuss their opinions on the meaning of collaboration in scientific research and (b) construct a Winogradsky column. While the column incubates between weeks 2 through 7, 212
Learning objectives Upon completion of this study, students will be able to:
PROCEDURE Materials Students receive a lab handout (Appendix 1). A table listing specific materials students will need for each week of the lab can be found in Appendix 2, Part 2. Lab coats, gloves, and goggles should be provided for the students when preparing and handling the contents of the columns. Instructors and students should be familiar with ASM’s Laboratory Safety Guidelines (http://www.asm.org/index. php/guidelines/safety-guidelines). To prepare the column, students will need a clear bottle to fill with soil and water. Carbon and/or sulfur sources (e.g., cellulose, calcium carbonate, corn starch, magnesium sulfate, calcium sulfate) should be added to the soil. The prepared column is to be incubated at room temperature in front of a light source.
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Opening the column and collecting the samples should be done in a chemical fume hood. Environmental samples are considered Biosafety Level 2 material, and students and instructors should follow proper handling and disposal protocol of biohazardous waste (Appendix 2). Since this activity calls for the use of DNA extraction kits, cloning kits, and sequencing, instructors should investigate the costs of such materials before deciding on assigning this experiment. Alternative modifications to the lab can be found in Appendix 2. Student instructions In the beginning of the semester, groups of four, consisting of two science and two non-science majors, are formed. The initial instructions for the lab are as follows. Discuss amongst yourselves what the phrase “collaboration in science” means to you. After you have done so, use the laboratory handout to prepare a Winogradsky column using local environmental samples and your choice of carbon and/or sulfur sources. Over the course of the semester, you will make observations based on what you see developing in the column. Your observations are expected to include writing and drawings depicting changes in color in the soil. After eight weeks, you will open the column and identify the enriched bacteria. Non-science majors will follow the chlorophyll identification protocol, while science majors will follow the 16S rRNA gene sequencing identification protocol. Once you have completed your identifications, each group will come back together and discuss their data. Faculty instructions After students have been placed into groups during the first week, instructors give an introduction to the assignment and the objectives of the exercise. During the eighth week, instructors will review the two methods of identification with all students. Instructors will emphasize that while students may be working in groups, each individual should answer all questions in their own words. Specific instructions for each week the groups meet can be found in Appendix 2. Evaluation. Majors and nonmajors prepare laboratory reports summarizing their findings (Appendix 1, Part 7). Instructors should grade their report based on the grading rubric provided in Appendix 2. Suggestions for determining student learning To determine whether learning objectives 1 and 2 were met, science majors and non-science majors from the fall 2013 and spring 2014 semesters were asked to complete two surveys. The first, two-question survey, was given at the beginning of the semester (Appendix 3). The material on Winogradsky columns was not previously covered in either class before the pre-survey. Students were expectVolume 16, Number 2
ed to rely on any previous science knowledge. Following completion of the laboratory exercise, a second, identical survey was given (Appendix 3). We analyzed the data using mixed methods consisting of both a qualitative review of participant responses and statistical analysis of the participant responses. Responses were qualitatively reviewed to pull out consistent themes and participant experiences. In addition, we coded survey responses into two dichotomous variables (0 or 1). A score of 0 indicated an incorrect, weak, or lack of response, and a score of 1 indicated a correct or strong answer. For question 1, strong answers were either detailed in their explanation and/or included themes and phrasing related to both “togetherness” and “across discipline” collaboration. For question 2, a score of 0 was equivalent to a simple answer or lack of an answer, whereas 1 was given for more in-depth responses. These could include “chlorophyll identification” or “16S rRNA gene sequencing.” Other alternative responses were also accepted. χ2-tests were utilized to determine whether there were any significant differences between the before and after responses. To determine whether learning objective 3 was met, we qualitatively analyzed the reports the students submitted at the end of the semester to see whether the students thought critically about the two methods they used and how they used those data to identify a microorganism that was enriched in the column. Evaluation of student perceptions. To determine the student’s perception of learning and their thoughts about the laboratory exercise, an additional survey was given to students at the end of the semester (Appendix 3). Sample data Over the course of the semester, groups should notice the development of colors in their Winogradsky columns due to the production of chlorophyll by phototrophic bacteria. These chlorophylls can be isolated using ethanol extraction and identified by spectroscopy (Fig. 1a). As seen in Figure 1a, the maximum peaks of absorption from chlorophyll extracted from soil 1 cm below the column surface occurs at 470 and 670 nm, which corresponds to chlorophyll a. Microorganisms containing chlorophyll a include green algae and cyanobacteria. Genomic DNA can also be isolated from this soil sample. Students performing PCR to amplify the 16S rRNA gene, using the primers listed in the procedure (Appendix 2), should generate a product of approximately 1500 bp (Fig. 1b). The 16S rRNA gene amplicon can then be ligated into the plasmid pGEM and sequenced. Sequencing of an amplicon from one such experiment indicated that the soil sample contained the cyanobacterium Rubidibacter lacunae. Collectively, these two results enabled students to discover that cyanobacteria are found 1 cm below the column surface. While preparing their lab report, students would realize that the location of cyanobacteria at the top of the column makes sense given that cyanobacteria perform
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FIGURE 1. Example of data collected during the experiment. Soil collected at 1 cm below the surface of the Winogradsky column was evenly divided. (a) An absorbance spectrum of chlorophyll extracted from the soil indicates the presence of chlorophyll a. (b) Extraction of DNA from the soil sample and subsequent PCR indicates successful amplification of the16S rRNA gene. Lane 1 = DNA ladder, Lane 2 = negative control sample where soil extracted DNA was not included in the reaction, Lane 3 = soil sample from 1 cm. DNA = deoxyribonucleic acid; PCR = polymerase chain reaction; rRNA = ribosomal ribonucleic acid.
oxygenic photosynthesis. Although this example shows that the two methods both identified cyanobacteria, it is possible for students to obtain different results using the two procedures.
DISCUSSION Evidence of student learning To assess objectives 1 and 2, we surveyed students before and after the completion of the laboratory exercise (Appendix 3). For our analysis of the first question (“Explain what the following phrase means to you: science is collaborative”), which addressed objective 1, we searched for answers that were similar to not only defining collaboration as “working together,” but rather “working together across different scientific fields” (Fig. 2a). It is interesting to note that our data suggest that students, regardless of major, already have a basic understanding of the term collaboration. In pre-surveys, the majority of students said that collaboration in science involved people working together, though few mentioned across disciplines in their answers. In the post-surveys, considerably more science majors and non-science majors answered this question incorporating both themes into their answers. These results suggest that this collaborative laboratory module successfully accomplished the first objective, and that in general, students had an improved understanding of scientific collaboration. Perhaps the increase in the common themes indicates that this real-life experience of mixing students from different disciplines can be beneficial in enhancing students’ learning experiences and scientific literacy. 214
For the second question, students were asked to name two methods for the identification of unknown bacteria. Correct answers were tallied (Fig. 2b). There was a statistically significant difference between science majors and non-science majors in their level of knowledge at the beginning of the semester (p < 0.0001). It is interesting to note that many of the science majors gave answers to the question using techniques that assumed the identification of pure cultures of bacteria. For example, several students suggested using Gram-staining and/or biochemical tests. Few gave answers for identifying non-culturable organisms. Many non-science majors were unable to answer the question. At the completion of the activity, there was no significant change in the majors’ ability to answer the question (p > 0.70). More majors did indicate using molecular biology techniques as a means of identification and were less reliant on pure-culture techniques. There was a significant increase in ability of the non-science majors to answer the question (p < 0.0001). Since students who dropped the course or did not complete the post-survey were from the science major group, we performed sensitivity analyses to test the robustness of our results. The sensitivity analyses showed that the post-survey relationship between major and nonmajors answers remains insignificant when accounting for the possible biasing effect of missing student data. In addition, the semester during which the course was taken (whether the course was taken in fall or spring) could be a potential confounding variable in the interpretation of the results. However, analysis showed that the students who took the courses in spring and fall were not significantly different in terms of class composition or in terms of the
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FIGURE 2. Assessment of learning objectives 1 and 2. Surveys were used to determine whether learning objectives 1 and 2 were met. Students were asked to (a) define scientific collaboration and (b) list methods of identifying bacteria. Results show percentage of students, by major, providing correct answers. Gray bars indicate pre-survey responses. Black bars indicate post-survey responses. *indicates a significant increase.
frequency in how they answered questions in relation to their major (p > 0.80). To assess objective 3, we used items 15 (“What type of photosynthetic microorganisms did you have?”) and 38 (“What organisms’ 16S rRNA gene did you amplify?”) from Appendix 1. We observed while reviewing their handouts and lab reports that all lab groups were able to identify enriched bacteria from the chlorophyll absorption spectra and from their BLAST analysis. Additionally, the laboratory reports could also be used to assess whether the exercise stimulated the students to think critically. Although this is not an explicit learning objective of the exercise, critical thinking is a general and universal goal of science education. The questions in the final laboratory reports were designed to guide the students and engage them in the process of critically analyzing their results by asking them to recall what nutrient supplies went into the columns, which organisms were identified, and what the metabolic characteristics of these organisms were. Students were then required to integrate these concepts through answering direct questions about the consistency of their results. Specifically, was there agreement between the two identification methods, and what was the likelihood of finding the identified organisms at the analyzed depth in the column? Instructors grading these reports observed that groups tried to reconcile differences between their data to develop an argument for what bacteria were present in the column, evidencing some degree of critical thinking about the limitations of each technique they used. For example, one group identified cyanobacteria at the bottom of their column. The group then indicated that cyanobacteria would probably not be found at the bottom of the column and Volume 16, Number 2
reasoned that even a low level of chlorophyll a at the bottom of the column could be detected using spectroscopy. Student opinions of the exercise. When asked about their opinions of collaboration and this experiment in particular, one science major answered that “two brains are better than one. More can be accomplished and discovered in a willing group.” Collaboration between majors was looked on favorably, with some non-science students looking forward to the opportunity to work with “real” science students. A non-science major commented, “I enjoyed working with other students who were far more familiar in a topic area than I was.” Qualitatively, some non-science major students appeared to have higher confidence in their own scientific abilities post activity. They realized their science ability was not much different than that of science majors. Many of the student opinions were positive, although some students were critical about the length of the lab.
CONCLUSION The lab exercise we prepared challenges both science majors and non-science majors and enhances scientific literacy. The activity allows students of any knowledge level to acquire unique environmental data by incorporating concepts from microbiology, genetics, and biochemistry. This results in the student gaining an insight into the wide diversity of microorganisms on our planet. Through this activity, students learn that they may need to rely on the expertise of their peers, and that together, they can answer scientific questions. According to our evaluation, the most significant increase in student knowledge was gained by the non-science majors. For
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non-science majors, making the exercise social and interactive improves the relevance of the coursework, meaning that the exercise can be an efficient and effective way to be engaged in science (5, 11). It also empowers students to cultivate a higher level of scientific literacy with assistance from their peers (6). Overall, our results suggest that students, regardless of major, learn about the collaborative nature of science while analyzing Winogradsky columns using this exercise.
SUPPLEMENTAL MATERIALS Appendix 1: Winogradsky column laboratory handout Appendix 2: Winogradsky column laboratory preparation directions & answer key Appendix 3: Survey distributed to assess student perceptions.
ACKNOWLEDGMENTS We thank Shantanu Bhatt, Catherine Sorace, and Joanna Huxster for allowing us to field test this laboratory exercise in their classes and for critical reading of this manuscript. We also thank Mackenzie Silvestri and Joseph Moran for their work in helping with data collection and analysis. A portion of this work has been supported through Saint Joseph’s University Summer Scholar’s Program. The authors declare that there are no conflicts of interest.
REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410.
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2. Anderson, D. C., and R. Hairston. 1999. The Winogradsky column and biofilms: models for teaching nutrient cycling and succession in an ecosystem. Am. Biol. Teach. 61:453–459. 3. Boomer, S., and K. Shipley. 2005. A laboratory class exploring and classifying anoxygenic phototrophic bacteria using culture-based approaches, microscopy and pigment analysis. American Society of Microbiology MicrobeLibrary. 4. Bruffee, K. A. 1994. The art of collaborative learning: making the most of knowledgeable peers. Change 26:39–44. 5. Hobson, A. 2001. Teaching relevant science for scientific literacy: adding cultural context to the sciences. J. Coll. Sci. Teach. 30:238–243. 6. Holt, C. E., P. Abramoff, L. V. Wilcox Jr., and D. L. Abell. 1969. Investigative laboratory programs in biology: a position paper of the commission on undergraduate education in the biological sciences. BioScience. 19:1104–1107. 7. National Research Council. 1999. Transforming undergraduate education in science, mathematics, engineering, and technology. The National Academies Press, Washington, DC. 8. Rutherford, F. J., and A. Ahlgren. 1991. Science for all Americans – AAAS project 2061. Oxford University Press, New York, NY. 9. Springer, L., M. E. Stanne, and S. S. Donovan. 1999. Effects of small-group learning on undergraduates in science, mathematics, engineering, and technology: a meta-analysis. Rev. Educ. Res. 69:21–51. 10. Sundberg, M. D., and M. L. Dini. 1993. Science majors vs non majors: is there a difference? J. Coll. Sci. Teach. 22:299–304. 11. Woodin, T., V. Carter, and L. Fletcher. 2010. Vision and change in biology undergraduate education, a call for action—initial responses. CBE Life Sci. Educ. 9:71–73.
Journal of Microbiology & Biology Education
Volume 16, Number 2
Curriculum
Discovery of the Collaborative Nature of Science with Undergraduate Science Majors and Non-Science Majors through the Identification of Microorganisms Enriched in Winogradsky Columns † Jasmine Ramirez1, Catalina Arango Pinedo1, and Brian M. Forster2* of Biology, Saint Joseph’s University, Philadelphia, PA 19131, 2College of Arts & Sciences, Saint Joseph’s University, Philadelphia, PA 19131 1Department
Today’s science classrooms are addressing the need for non-scientists to become scientifically literate. A key aspect includes the recognition of science as a process for discovery. This process relies upon interdisciplinary collaboration. We designed a semester-long collaborative exercise that allows science majors taking a general microbiology course and non-science majors taking an introductory environmental science course to experience collaboration in science by combining their differing skill sets to identify microorganisms enriched in Winogradsky columns. These columns are self-sufficient ecosystems that allow researchers to study bacterial populations under specified environmental conditions. Non-science majors identified phototrophic bacteria enriched in the column by analyzing the signature chlorophyll absorption spectra whereas science majors used 16S rRNA gene sequencing to identify the general bacterial diversity. Students then compiled their results and worked together to generate lab reports with their final conclusions identifying the microorganisms present in their column. Surveys and lab reports were utilized to evaluate the learning objectives of this activity. In pre-surveys, nonmajors’ and majors’ answers diverged considerably, with majors providing responses that were more accurate and more in line with the working definition of collaboration. In post-surveys, the answers between majors and nonmajors converged, with both groups providing accurate responses. Lab reports showed that students were able to successfully identify bacteria present in the columns. These results demonstrate that laboratory exercises designed to group students across disciplinary lines can be an important tool in promoting science education across disciplines.
INTRODUCTION With the ongoing discussion of American scientific illiteracy in mind, we have designed a semester-long experiment that promotes the collaborative nature of science as a means of enhancing science education. Students have the misconception that today’s scientists are highly specialized workers in a specific area. Rather, scientists collaborate by exchanging ideas and seek the opinions of others outside their field to answer a question. Recommendations on learning and teaching suggest that teachers should promote collaboration through group work, and that there should be an emphasis on the idea that science is a collection of different scientific fields or content disciplines (8). Therefore, it is important to have students participate in multi-field experiments. Incorporating collaboration in the classroom can benefit students, especially in classes that have both science *Corresponding author. Mailing address: 5600 City Avenue, 112 Connelly Hall, Philadelphia, PA 19131. Phone: 610-660-3188. E-mail: [email protected]. †Supplemental materials available at http://jmbe.asm.org
and non-science major students (4, 9, 10). The National Research Council has reported that STEM students quickly specialize in one specific discipline while other students take a year or less of STEM courses (7). To address this, science curricula should seek to foster collaborative learning so that both STEM and non-STEM students can have their learning experience enhanced and understand the benefits of collaboration in the scientific process. This paper describes a semester-long laboratory exercise where science majors and non-science majors collaborate to construct and analyze Winogradsky columns. This ecosystem provides a convenient means for studying the growth requirements of bacteria (2). Columns are packed with soil and water. The soil can be amended with cellulose and additional carbon (organic and/or inorganic) and sulfur sources to enrich for various types of autotrophic and heterotrophic bacteria. In the column, the organic material at the bottom of the column is used up, resulting in depleted O2 at the bottom of the column and higher levels of O2 at the top. The anaerobes that are now selected for at the bottom of the column produce carbon by-products which are then used by sulfate-utilizing bacteria to produce H2S.
©2015 Author(s). Published by the American Society for Microbiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
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This results in high concentrations of H2S at the bottom of the column and lower levels at the top. The H2S is then available for cellular respiration or as an electron source for photosynthesis. The column promotes the growth of different bacterial types at various depths corresponding to O2 and H2S concentrations. The columns are subsequently incubated in front of a light source to enrich for phototrophic bacteria for approximately six to eight weeks. This exercise teaches students two different methods of identifying bacterial species in the column. One method is extracting chlorophyll and analyzing its absorbance spectra. This is a very broad method for identification and is restricted to the identification of phototrophs, both bacteria and eukaryotes. The second method is sequencing the gene that encodes the 16S ribosomal ribonucleic acid (rRNA) found in the small ribosomal unit of bacteria and identifying a spectrum of bacterial species using basic local alignment search tool (BLAST) analysis (1). A previous exercise has been published which describes the use of the chlorophyll identification as a means of identifying enriched anoxic phototrophic microorganisms from environmental samples (3). In that study, pure cultures of oxygenic phototrophic bacteria were utilized. Following the chlorophyll identification of the samples, students used microscopy to visualize microorganisms from their enrichments. In this lab exercise, we allow students to enrich for both oxic and anoxic microorganisms and have groups supplement their chlorophyll identification with 16S rRNA gene analysis.
instructors should plan to have their students work on other laboratory activities that may or may not be related to this activity. Groups should independently make their observations once a week. Observations are expected to include writing and drawings depicting changes in soil color. During the incubation period between weeks 2 through 7, science majors should learn about deoxyribonucleic acid (DNA) isolation, polymerase chain reaction (PCR), and 16S rRNA gene sequencing. Non-science majors should learn about chlorophyll and phototrophic bacteria. In the eighth week, groups meet to discuss what they have learned and open their columns. Non-science majors identify phototrophic microorganisms through analysis of the chlorophyll absorbance spectra. This procedure will take approximately one hour. Science majors isolate genomic DNA from the soil and identify bacteria by 16S rRNA gene sequencing. This will take three lab sessions of three hours each. During their last meeting of the semester, group members discuss their results and prepare lab reports (Appendices 1 and 2). While majors and nonmajors collaborate on their data and discuss with one another the strengths and weaknesses of each technique used, they each prepare separate reports and are graded on expectations commensurate with their level of scientific knowledge.
Intended audience/Prerequisite student knowledge
1. D efine the term collaboration as it applies to the nature of science. 2. Name two different techniques for identifying microorganisms. 3. Use absorbance spectra of (bacterio)chlorophyll and 16S rRNA gene sequencing to identify microorganisms that have been enriched in a Winogradsky column.
This activity was designed for groups consisting of biology majors (general microbiology students) and non-science majors (introductory environmental science students). Both groups of students should have a basic understanding of the fact that bacteria can be found in different environments depending upon their nutritional and gas requirements in order to grow and of the process of photosynthesis. They should also be familiar with aseptic technique. While there is no additional prerequisite knowledge for non-science majors, science majors should be familiar with what it means for a bacterium to be defined as a phototroph and with basic concepts of genetics. This type of exercise may be appropriate for science lab-based courses of any level, including microbiology classes. This activity can also be used in large class settings, with modifications described in Appendix 2. Learning time This activity has seven parts spanning an entire academic semester. Science major and non-science major students meet three times in the semester. Labs meet for three hours, once a week. In the first week, the students (a) discuss their opinions on the meaning of collaboration in scientific research and (b) construct a Winogradsky column. While the column incubates between weeks 2 through 7, 212
Learning objectives Upon completion of this study, students will be able to:
PROCEDURE Materials Students receive a lab handout (Appendix 1). A table listing specific materials students will need for each week of the lab can be found in Appendix 2, Part 2. Lab coats, gloves, and goggles should be provided for the students when preparing and handling the contents of the columns. Instructors and students should be familiar with ASM’s Laboratory Safety Guidelines (http://www.asm.org/index. php/guidelines/safety-guidelines). To prepare the column, students will need a clear bottle to fill with soil and water. Carbon and/or sulfur sources (e.g., cellulose, calcium carbonate, corn starch, magnesium sulfate, calcium sulfate) should be added to the soil. The prepared column is to be incubated at room temperature in front of a light source.
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Opening the column and collecting the samples should be done in a chemical fume hood. Environmental samples are considered Biosafety Level 2 material, and students and instructors should follow proper handling and disposal protocol of biohazardous waste (Appendix 2). Since this activity calls for the use of DNA extraction kits, cloning kits, and sequencing, instructors should investigate the costs of such materials before deciding on assigning this experiment. Alternative modifications to the lab can be found in Appendix 2. Student instructions In the beginning of the semester, groups of four, consisting of two science and two non-science majors, are formed. The initial instructions for the lab are as follows. Discuss amongst yourselves what the phrase “collaboration in science” means to you. After you have done so, use the laboratory handout to prepare a Winogradsky column using local environmental samples and your choice of carbon and/or sulfur sources. Over the course of the semester, you will make observations based on what you see developing in the column. Your observations are expected to include writing and drawings depicting changes in color in the soil. After eight weeks, you will open the column and identify the enriched bacteria. Non-science majors will follow the chlorophyll identification protocol, while science majors will follow the 16S rRNA gene sequencing identification protocol. Once you have completed your identifications, each group will come back together and discuss their data. Faculty instructions After students have been placed into groups during the first week, instructors give an introduction to the assignment and the objectives of the exercise. During the eighth week, instructors will review the two methods of identification with all students. Instructors will emphasize that while students may be working in groups, each individual should answer all questions in their own words. Specific instructions for each week the groups meet can be found in Appendix 2. Evaluation. Majors and nonmajors prepare laboratory reports summarizing their findings (Appendix 1, Part 7). Instructors should grade their report based on the grading rubric provided in Appendix 2. Suggestions for determining student learning To determine whether learning objectives 1 and 2 were met, science majors and non-science majors from the fall 2013 and spring 2014 semesters were asked to complete two surveys. The first, two-question survey, was given at the beginning of the semester (Appendix 3). The material on Winogradsky columns was not previously covered in either class before the pre-survey. Students were expectVolume 16, Number 2
ed to rely on any previous science knowledge. Following completion of the laboratory exercise, a second, identical survey was given (Appendix 3). We analyzed the data using mixed methods consisting of both a qualitative review of participant responses and statistical analysis of the participant responses. Responses were qualitatively reviewed to pull out consistent themes and participant experiences. In addition, we coded survey responses into two dichotomous variables (0 or 1). A score of 0 indicated an incorrect, weak, or lack of response, and a score of 1 indicated a correct or strong answer. For question 1, strong answers were either detailed in their explanation and/or included themes and phrasing related to both “togetherness” and “across discipline” collaboration. For question 2, a score of 0 was equivalent to a simple answer or lack of an answer, whereas 1 was given for more in-depth responses. These could include “chlorophyll identification” or “16S rRNA gene sequencing.” Other alternative responses were also accepted. χ2-tests were utilized to determine whether there were any significant differences between the before and after responses. To determine whether learning objective 3 was met, we qualitatively analyzed the reports the students submitted at the end of the semester to see whether the students thought critically about the two methods they used and how they used those data to identify a microorganism that was enriched in the column. Evaluation of student perceptions. To determine the student’s perception of learning and their thoughts about the laboratory exercise, an additional survey was given to students at the end of the semester (Appendix 3). Sample data Over the course of the semester, groups should notice the development of colors in their Winogradsky columns due to the production of chlorophyll by phototrophic bacteria. These chlorophylls can be isolated using ethanol extraction and identified by spectroscopy (Fig. 1a). As seen in Figure 1a, the maximum peaks of absorption from chlorophyll extracted from soil 1 cm below the column surface occurs at 470 and 670 nm, which corresponds to chlorophyll a. Microorganisms containing chlorophyll a include green algae and cyanobacteria. Genomic DNA can also be isolated from this soil sample. Students performing PCR to amplify the 16S rRNA gene, using the primers listed in the procedure (Appendix 2), should generate a product of approximately 1500 bp (Fig. 1b). The 16S rRNA gene amplicon can then be ligated into the plasmid pGEM and sequenced. Sequencing of an amplicon from one such experiment indicated that the soil sample contained the cyanobacterium Rubidibacter lacunae. Collectively, these two results enabled students to discover that cyanobacteria are found 1 cm below the column surface. While preparing their lab report, students would realize that the location of cyanobacteria at the top of the column makes sense given that cyanobacteria perform
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FIGURE 1. Example of data collected during the experiment. Soil collected at 1 cm below the surface of the Winogradsky column was evenly divided. (a) An absorbance spectrum of chlorophyll extracted from the soil indicates the presence of chlorophyll a. (b) Extraction of DNA from the soil sample and subsequent PCR indicates successful amplification of the16S rRNA gene. Lane 1 = DNA ladder, Lane 2 = negative control sample where soil extracted DNA was not included in the reaction, Lane 3 = soil sample from 1 cm. DNA = deoxyribonucleic acid; PCR = polymerase chain reaction; rRNA = ribosomal ribonucleic acid.
oxygenic photosynthesis. Although this example shows that the two methods both identified cyanobacteria, it is possible for students to obtain different results using the two procedures.
DISCUSSION Evidence of student learning To assess objectives 1 and 2, we surveyed students before and after the completion of the laboratory exercise (Appendix 3). For our analysis of the first question (“Explain what the following phrase means to you: science is collaborative”), which addressed objective 1, we searched for answers that were similar to not only defining collaboration as “working together,” but rather “working together across different scientific fields” (Fig. 2a). It is interesting to note that our data suggest that students, regardless of major, already have a basic understanding of the term collaboration. In pre-surveys, the majority of students said that collaboration in science involved people working together, though few mentioned across disciplines in their answers. In the post-surveys, considerably more science majors and non-science majors answered this question incorporating both themes into their answers. These results suggest that this collaborative laboratory module successfully accomplished the first objective, and that in general, students had an improved understanding of scientific collaboration. Perhaps the increase in the common themes indicates that this real-life experience of mixing students from different disciplines can be beneficial in enhancing students’ learning experiences and scientific literacy. 214
For the second question, students were asked to name two methods for the identification of unknown bacteria. Correct answers were tallied (Fig. 2b). There was a statistically significant difference between science majors and non-science majors in their level of knowledge at the beginning of the semester (p < 0.0001). It is interesting to note that many of the science majors gave answers to the question using techniques that assumed the identification of pure cultures of bacteria. For example, several students suggested using Gram-staining and/or biochemical tests. Few gave answers for identifying non-culturable organisms. Many non-science majors were unable to answer the question. At the completion of the activity, there was no significant change in the majors’ ability to answer the question (p > 0.70). More majors did indicate using molecular biology techniques as a means of identification and were less reliant on pure-culture techniques. There was a significant increase in ability of the non-science majors to answer the question (p < 0.0001). Since students who dropped the course or did not complete the post-survey were from the science major group, we performed sensitivity analyses to test the robustness of our results. The sensitivity analyses showed that the post-survey relationship between major and nonmajors answers remains insignificant when accounting for the possible biasing effect of missing student data. In addition, the semester during which the course was taken (whether the course was taken in fall or spring) could be a potential confounding variable in the interpretation of the results. However, analysis showed that the students who took the courses in spring and fall were not significantly different in terms of class composition or in terms of the
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FIGURE 2. Assessment of learning objectives 1 and 2. Surveys were used to determine whether learning objectives 1 and 2 were met. Students were asked to (a) define scientific collaboration and (b) list methods of identifying bacteria. Results show percentage of students, by major, providing correct answers. Gray bars indicate pre-survey responses. Black bars indicate post-survey responses. *indicates a significant increase.
frequency in how they answered questions in relation to their major (p > 0.80). To assess objective 3, we used items 15 (“What type of photosynthetic microorganisms did you have?”) and 38 (“What organisms’ 16S rRNA gene did you amplify?”) from Appendix 1. We observed while reviewing their handouts and lab reports that all lab groups were able to identify enriched bacteria from the chlorophyll absorption spectra and from their BLAST analysis. Additionally, the laboratory reports could also be used to assess whether the exercise stimulated the students to think critically. Although this is not an explicit learning objective of the exercise, critical thinking is a general and universal goal of science education. The questions in the final laboratory reports were designed to guide the students and engage them in the process of critically analyzing their results by asking them to recall what nutrient supplies went into the columns, which organisms were identified, and what the metabolic characteristics of these organisms were. Students were then required to integrate these concepts through answering direct questions about the consistency of their results. Specifically, was there agreement between the two identification methods, and what was the likelihood of finding the identified organisms at the analyzed depth in the column? Instructors grading these reports observed that groups tried to reconcile differences between their data to develop an argument for what bacteria were present in the column, evidencing some degree of critical thinking about the limitations of each technique they used. For example, one group identified cyanobacteria at the bottom of their column. The group then indicated that cyanobacteria would probably not be found at the bottom of the column and Volume 16, Number 2
reasoned that even a low level of chlorophyll a at the bottom of the column could be detected using spectroscopy. Student opinions of the exercise. When asked about their opinions of collaboration and this experiment in particular, one science major answered that “two brains are better than one. More can be accomplished and discovered in a willing group.” Collaboration between majors was looked on favorably, with some non-science students looking forward to the opportunity to work with “real” science students. A non-science major commented, “I enjoyed working with other students who were far more familiar in a topic area than I was.” Qualitatively, some non-science major students appeared to have higher confidence in their own scientific abilities post activity. They realized their science ability was not much different than that of science majors. Many of the student opinions were positive, although some students were critical about the length of the lab.
CONCLUSION The lab exercise we prepared challenges both science majors and non-science majors and enhances scientific literacy. The activity allows students of any knowledge level to acquire unique environmental data by incorporating concepts from microbiology, genetics, and biochemistry. This results in the student gaining an insight into the wide diversity of microorganisms on our planet. Through this activity, students learn that they may need to rely on the expertise of their peers, and that together, they can answer scientific questions. According to our evaluation, the most significant increase in student knowledge was gained by the non-science majors. For
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non-science majors, making the exercise social and interactive improves the relevance of the coursework, meaning that the exercise can be an efficient and effective way to be engaged in science (5, 11). It also empowers students to cultivate a higher level of scientific literacy with assistance from their peers (6). Overall, our results suggest that students, regardless of major, learn about the collaborative nature of science while analyzing Winogradsky columns using this exercise.
SUPPLEMENTAL MATERIALS Appendix 1: Winogradsky column laboratory handout Appendix 2: Winogradsky column laboratory preparation directions & answer key Appendix 3: Survey distributed to assess student perceptions.
ACKNOWLEDGMENTS We thank Shantanu Bhatt, Catherine Sorace, and Joanna Huxster for allowing us to field test this laboratory exercise in their classes and for critical reading of this manuscript. We also thank Mackenzie Silvestri and Joseph Moran for their work in helping with data collection and analysis. A portion of this work has been supported through Saint Joseph’s University Summer Scholar’s Program. The authors declare that there are no conflicts of interest.
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2. Anderson, D. C., and R. Hairston. 1999. The Winogradsky column and biofilms: models for teaching nutrient cycling and succession in an ecosystem. Am. Biol. Teach. 61:453–459. 3. Boomer, S., and K. Shipley. 2005. A laboratory class exploring and classifying anoxygenic phototrophic bacteria using culture-based approaches, microscopy and pigment analysis. American Society of Microbiology MicrobeLibrary. 4. Bruffee, K. A. 1994. The art of collaborative learning: making the most of knowledgeable peers. Change 26:39–44. 5. Hobson, A. 2001. Teaching relevant science for scientific literacy: adding cultural context to the sciences. J. Coll. Sci. Teach. 30:238–243. 6. Holt, C. E., P. Abramoff, L. V. Wilcox Jr., and D. L. Abell. 1969. Investigative laboratory programs in biology: a position paper of the commission on undergraduate education in the biological sciences. BioScience. 19:1104–1107. 7. National Research Council. 1999. Transforming undergraduate education in science, mathematics, engineering, and technology. The National Academies Press, Washington, DC. 8. Rutherford, F. J., and A. Ahlgren. 1991. Science for all Americans – AAAS project 2061. Oxford University Press, New York, NY. 9. Springer, L., M. E. Stanne, and S. S. Donovan. 1999. Effects of small-group learning on undergraduates in science, mathematics, engineering, and technology: a meta-analysis. Rev. Educ. Res. 69:21–51. 10. Sundberg, M. D., and M. L. Dini. 1993. Science majors vs non majors: is there a difference? J. Coll. Sci. Teach. 22:299–304. 11. Woodin, T., V. Carter, and L. Fletcher. 2010. Vision and change in biology undergraduate education, a call for action—initial responses. CBE Life Sci. Educ. 9:71–73.
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