Hormones and antibiotics in nature: a laboratory module designed to broaden undergraduate perspectives on typically human-centered topics.

Bringing discovery-based research into undergraduate laboratory courses increases student motivation and learning gains over traditional exercises that merely teach technique or demonstrate well-documented phenomena. Laboratory experiences are further enhanced when they are designed to challenge student perspectives on topics relevant to their lives. To this end, a laboratory module on antibiotics and hormones, which are generally discussed in the context of human health, was developed for students to explore the multifaceted roles of antibiotics and hormones in nature (e.g. interspecies communication) via reading primary scientific literature and performing discovery-based experiments. The main objective of this module was to increase the general biological literacy of students as determined by their ability to connect the Five Core Concepts of Biological Literacy (American Association for the Advancement of Science, Vision and Change in Undergraduate Education: A Call to Action, 2011) to the topics "hormones" and "antibiotics" in pre- and postmodule surveys. After discussing unpublished research findings, cell biology students performed experiments demonstrating that: 1) fungi may promote fern growth via hormone production, 2) novel bacterial isolates in the genus Streptomyces produce antifungal compounds, and 3) subinhibitory antibiotic concentrations may enhance soil bacterial growth. The third finding provided evidence supporting a hypothesis framed in a scientific article that students read and discussed. Student perspectives on premodule surveys focused on roles of hormones and antibiotics in the human body (e.g. development, fighting infection), but their broadened postmodule perspectives encompassed the roles of these molecules in organismal communication and possibly the evolution of multicellularity.

INTRODUCTION

Bringing discovery-based research into undergraduate biology laboratory courses increases student motivation and learning gains over traditional exercises that merely teach technique or demonstrate well-documented phenomena. Learning technique and the scientific method in the context of real scientific research provides connectivity and purpose in laboratory exercises, which students may otherwise perceive as a list of graded tasks (1, 15, 19).

Laboratory course experiences can be further enhanced and made more memorable when hands-on experiments revolve around topics that are relevant to students’ lives, especially when they challenge perspectives that have been ingrained in students through previous course-work and/or life experiences (6). In efforts to increase biological literacy in general, creating such experiences becomes critical when students are enrolled in narrow, occupation-specific curricula. The curriculum supporting career tracks in health professions (e.g. nursing, pharmacy) can be particularly focused, emphasizing human-centered topics to the detriment of teaching broader concepts in biology and the human position within an ecosystem (3). Understanding that ecosystem and human health are intimately intertwined is crucial for students to develop problem-solving skills for the future (20). However, this is not well conveyed in traditional university courses that focus very narrowly within a subdiscipline of the biological sciences (e.g. ecology, molecular biology).

Hormones and molecules that we call antibiotics are most commonly discussed within the context of human health, but they have been utilized as chemical signaling molecules in microbe-microbe, plant-microbe and plant-plant interactions throughout the course of evolution (18, 9). Investigating how these communication pathways work could potentially provide insights into solving human food and drug crises. For instance, as new agricultural strategies are being developed to feed the world’s burgeoning population, there is increased interest in looking to nature for solutions, such as microbial production of plant growth-promoting hormones (1, 8). As researchers work toward engineering microbial populations for agricultural use, there is keen interest in understanding how microbes interact and communicate amongst themselves. This is also true in the case of engineering microbial communities (e.g. probiotics) to fight disease (5); this necessitates knowing how microbes respond and develop resistance to various treatments like antibiotic consumption. In light of this, Davies (9), provides an important perspective on this area of research, pointing out that microbes have been producing molecules that humans call “antibiotics” for ages, even though they may not function as chemical weapons in nature. Davies (9) and others (21) propose that “antibiotics” exhibit hormesis, having negative or positive impacts on organism growth depending on the concentration of “antibiotics” in the environment. For instance, “antibiotics” may actually function as cell-signaling molecules in nature where they are present at subtherapeutic concentrations and may have even played a role in the evolution of multicellularity. Understanding how “antibiotics” may promote microbial cooperation could provide insights for fighting infection.

This laboratory module focuses on increasing undergraduate biological literacy and broadening student perspectives on antibiotics and hormones to include their multifaceted roles in nature. Students are provided with background information on these topics via interactive lectures, readings in the primary scientific literature, and class discussions. Students are guided through the process of critically analyzing scientific literature in conjunction with unpublished data from a research laboratory at the University to design the next experiment. Groups of four students set up three experiments to examine the possibility that 1) fungi produce hormones that enhance fern growth in a bioassay designed to mimic macronutrient conditions found in nature, 2) subinhibitory concentrations of antibiotics enhance the growth of soil bacteria, and 3) novel bacterial isolates in a collection of Streptomyces species produce anti-fungal compounds. These experiments, which take place at the beginning of the semester, provide first-hand experience with the subject matter and generate discussions regarding how organisms interact and how their needs to acquire basic chemical building blocks for the cell can drive the evolution of such interactions (e.g. competition or cooperation). Of the Five Core Concepts for Biological Literacy (1), this targets “Systems, Pathways and Transformations of Energy and Matter” as well as “Evolution” (Table 1). Discussions of antibiotics and hormones and how they impact organism growth and behavior target the “Structure and Function” and “Information Flow, Exchange and Storage” concepts, which are discussed intently in the following segment of the course (DNA replication and gene expression).

TABLE 1.

The Five Core Concepts for Biological Literacy (1).

Concept	Description
Evolution	The diversity of life evolved over time by processes of mutation, selection, and genetic change.
Structure & Function	Basic units of structure define the function of all living things.
Information Flow, Exchange & Storage	The growth and behavior of organisms are activated through the expression of genetic information in context.
Pathways & Transformations of Energy & Matter	Biological systems grow and change by processes based upon chemical transformation pathways and are governed by the laws of thermodynamics.
Systems	Living systems are interconnected and interacting.

Learning objectives

By guiding students through the process of questioning published scientific results, examining unpublished experimental data and designing discovery-based experiments to explore the roles of hormones and antibiotics in nature (e.g. interspecies communication), the overall objective of this module is to broaden student perspectives and increase their general biological literacy. Accomplishment of this objective will be determined by the ability of students to connect the Five Core Concepts for Biological Literacy (1) to the topics “hormones” and “antibiotics” in pre- and postmodule surveys. Specifically, surveys will be used to determine: 1) whether or not students can connect the topics “hormones” and “antibiotics” to an increased number of the Five Core Concepts in postmodule surveys than in premodule surveys, 2) whether or not students can describe these connections in greater depth (i.e. more detailed explanations and/ or referral to specific examples from module content) in postmodule surveys than in premodule surveys.

Additionally, with regard to the specific content of the module, students will be able to do the following in postmodule surveys:

Describe hormesis as it relates to antibiotics and how the growth of microorganisms may respond.

Describe hormones and antibiotics as signaling molecules that can be used in interspecies communication.

Describe the importance of basic research on chemical communication in fueling applied research.

Comparison of pre- and postmodule surveys indicated that the above objectives were accomplished at the same time the students conducted experiments that provided novel scientific results for ongoing research projects at Idaho State University. Experimental results provided supporting evidence for a hypothesis that the students read about in the primary literature (9) suggesting that subinhibitory concentrations of antibiotics enhance microbial growth. Student observations also demonstrated that fungi may produce hormones that enhance fern growth even when macronutrient concentrations in typical bioassays are reduced 500-fold to mimic concentrations found in nature. Additionally, students observed that fern growth in the absence of fungi was not dramatically enhanced on full-strength growth medium relative to 500-fold diluted growth medium, indicating that the presence of large amounts of nutrients does not always lead to growing larger plants. Students were able to extrapolate this finding to agricultural settings and understand how, in many cases, fertilizer application is unnecessarily high. This was borne out in class discussions about not only the concentration of nutrients that plants need but the ratio in which they are needed for growth; calculations of nitrogen:phosphorus ratios in the growth medium revealed that phosphorus could be a limiting nutrient in this medium. Lastly, students determined that novel Streptomyces species could inhibit fungal growth. This showed students the value of microbial bioprospecting efforts in applied research (e.g. drug development).

This module relates to a major challenge area identified in a 2009 report by the National Research Council, A New Biology for the 21 which is to “understand and sustain ecosystem function and biodiversity in the face of rapid change” (14). This module also embraces the following action items within the AAAS Vision and Change report (1): aligning assessment with understanding core concepts, making biology content relevant by presenting it in the context of real-world problems (e.g. food and drug crises), stimulating student curiosity for learning about the natural world and engaging students as active participants in discovery-based research regardless of their proposed major or career track.

Intended audience/prerequisite student knowledge

The module described within was designed for a Cell Biology Laboratory course (BIOL 2207) at Idaho State University (ISU), with a maximum enrollment of 72 students (24 students in three sections) that is offered every spring semester (16 weeks). Prerequisites for this course are the Introductory Biology series (BIOL 1101 and 1102) and Introduction to General Chemistry I (CHEM 1111); Introduction to Organic and Biochemistry (CHEM 1112) is a co-requisite. Students are expected to have prior knowledge of cellular chemistry and macronutrients required for growth, how to calculate molarity, and the central dogma, as well as basic ideas regarding the use of antibiotics to kill pathogens and hormonal control of organismal growth. This knowledge will aid students in understanding the scientific literature that is read and discussed as background information for the experiments. While many of the students in BIOL 2207 have already taken General Microbiology and have experience with techniques like spread plating and using micropipettors, this experience is not necessary if the instructor provides information on the purpose of the technique and demonstrates how it is properly performed. It is assumed that students have experience with reading primary scientific literature in previous courses and are familiar with the scientific method. As discussed below, this module could be modified to provide discovery-based experiences in upper division courses.

Learning time

BIOL 2207 at ISU consists of one 165-minute time slot per week. The course is divided into four segments (three to five weeks each) and a different module is taught during each segment. Each module, led by one instructor, contains a series of laboratory experiments and discussions of primary scientific literature centered around a theme from his or her area of expertise that relates to core concepts being presented in the lecture course at that time.

Table 2 contains an outline of module activities that took place during three lab periods (three weeks). During the first two weeks of the course, students were engaged in interactive lectures and class discussions regarding hormones and antibiotics and the roles that they play in microbe-microbe, plant-microbe and plant-plant interactions as well as the applications of basic research to potentially solving food and drug crises. Interactive lectures (Outlined in Appendices 2 and 5) present information from several sources (2, 4, 7, 8, 10, 13, 16, 17) in addition to Davies (9) and Contreras-Cornejo (8), which the students read. Class discussions and group work leading up to the experiments include developing hypotheses and calculating macronutrient concentrations and stoichiometries in typical plant-microbe bioassay conditions (Appendix 3).

TABLE 2.

Outline of module activities and corresponding resources for each of three 165-minute class periods held one week apart.

Class Period	Activity Type	Description	Resources
1	Writing/assessment	Premodule survey	Appendix 1
	Critical thinking	Interactive instructor presentation (hormones in plant and microbial communication)	Appendix 2
	Critical thinking	Reading & discussion of Contreras-Cornejo et al. (8) abstract, introduction, select figures and methods	Contreras-Cornejo et al. 2009 (8)
	Quantitative problem solving and analysis	Calculation of macronutrient molar concentration & stoichiometry in bioassay conditions vs. nature	Appendix 3
	Critical thinking	Interactive instructor presentation (preliminary experimental data)	Appendix 2; (Boydstun et al., unpublished)
	Group work	Experiment 1: Could a fungus promote fern growth by producing phytohormones?	Appendix 4
2	Assessment	Quiz over Davies, 2006 (9)	Davies, 2006 (9) Appendix 6
	Critical thinking	Class discussion about Davies, 2006 (9) and hypothesis generation	Appendix 5
	Group work	Experiment 2: What is the impact of subinhibitory antibiotic concentrations on soil bacterial growth?	Appendix 4
	Critical thinking	Interactive instructor presentation (Streptomyces development & antibiotic production)	Appendix 5
	Group work	Experiment 3: Do novel Streptomyces isolated from the atmosphere produce antifungal compounds?	Appendix 4
3	Group work and data analysis	Data collection	Appendix 4
	Writing/assessment	Postmodule survey	Appendix 1

In weeks one and two, students set up three experiments (Table 2). Experiment 1 examines whether a fungus may promote fern growth (Ceratopteris richardii) via hormone production. Before setting up this experiment, students are presented with unpublished experimental data from a research laboratory at ISU (Boydstun et al., unpublished data) demonstrating that ferns grew larger when co-cultured with a fungus (Alternaria sp. BR; Appendix 2). In these preliminary experiments, ferns and fungus were in direct contact with each other. In order to determine whether this result was due to chemical signaling between the organisms rather than physical contact, students performed experiments in which ferns were grown on top of polycarbonate membranes, which were placed over fungi growing on agar medium; the membranes physically separated the two organisms, but allowed for potential passing of chemical signals between them. Proper controls ensured that the membrane did not inhibit fern spore germination and growth. Three types of agar medium were utilized in this experiment: basal salts medium (BSM) represented the macronutrient-rich media typically utilized in bioassays, and 250- and 500-fold dilutions of BSM mimicked macronutrient concentrations found in nature (Appendix 4).

Experiment 2 tested the hypothesis that subinhibitory concentrations of antibiotics enhance soil bacterial growth. Before performing this experiment, students developed hypotheses regarding what they would observe if they cultivated soil bacteria on growth medium containing 0, 0.05, 0.5, 5 or 50 μg/mL of various antibiotics. Six groups of students in each laboratory section performed a spread plating experiment utilizing one of six possible antibiotics (Appendix 4).

Experiment 3 aimed to determine whether novel Streptomyces species could produce antifungal compounds (Appendix 4). The collection of Streptomyces species was previously isolated from outdoor air as part of a National Science Foundation Project to characterize the biodiversity of air and rain (Weber et al., unpublished).

In week three, students collected data on all three experiments and discussed them as a class. The module concluded with students taking a postmodule survey. They were provided with the material that they wrote on their premodule surveys and asked if they could augment what they had written previously based on their experience with the material and activities in the laboratory module.

PROCEDURE

Materials and student instructions

Complete equipment and supply lists for all experiments are provided in Appendix 4. During class, students are provided with diagrams and instructions displayed in the room on PowerPoint slides (Appendices 2 and 5) and the instructor provides verbal explanations and demonstrates the procedures (Appendix 4). This approach saves paper and captures student attention better than having them distracted by written protocols on paper while the instructor is demonstrating technique. However, written step-by-step procedures for students are available in Appendix 4 for printing and distribution if desired.

Briefly, to set up Experiment 1, each student group received four agar plates, two with fungi and two without fungi (all plates contained either BSM, 1/500 BSM, or 1/250 BSM). Students used presterilized tweezers to place polycarbonate membranes on one plate with fungi and one without fungi. A cotton swab was used to apply spores to the top of both membranes and to the plate lacking a membrane or fungi. Plates containing fern spores were incubated under grow lights for two weeks. In week three, students observed plates using a dissecting microscope and verbally shared observations with the entire class.

Students set up Experiment 2 by serially diluting a soil bacterial extract (5 g of soil in 25 mL of 1× phosphate buffer) to 10−5. Each student group spread 50 μL of the 10−5 dilution onto King’s B agar medium plates containing 0, 0.05, 0.5, 5 or 50 μg/mL of one of six different antibiotics. Plates were parafilmed and stored at room temperature for one week. In week three, the number of colonies was counted and recorded and graphed on a class spreadsheet.

For Experiment 3, each student group received a King’s B medium plate that contained a different Streptomyces species spotted in the center (spot plate). Each group aseptically transferred a cube of agar cut from a potato dextrose agar plate containing the test fungus (Alternaria sp. BR) to the edge of the agar in the spot plate. Plates were sealed with parafilm and incubated at room temperature for one week. In week three, plates were observed for zones of clearing indicating that the bacterial isolate was producing an antifungal compound.

Faculty instructions

Detailed instructor protocols and growth medium and reagent recipes are provided in Appendix 4; lecture and discussion outlines are provided in Appendices 2 and 5. Briefly, in week one, the instructor introduces plant hormones and leads the students in a discussion of Contreras-Cornejo et al. (8). The instructor also demonstrates procedures for setting up Experiment 1. In week two, the instructor administers a quiz and leads a discussion of Davies (9), as well as antibiotic production by Streptomyces. The instructor also demonstrates proper technique and safety for serial dilution and spread plating as well as manipulating fungal cultures on agar medium. In week three, the instructor guides students through observations, data collection, and interpretation of the results.

Experiment preparation will vary with class size. All of the growth media can be prepared a few weeks in advance and kept in a refrigerator. Cultures of the fungus used in the field test of this module (Alternaria sp. BR) required only about two days to grow on potato dextrose agar (PDA), but about a week on any variation of BSM medium. Streptomyces cultures require 3–5 days to grow on King’s B medium. A detailed timeline of lab preparation is included in Appendix 4.

Suggestions for determining student learning

Student learning was assessed through comparison of pre- and postmodule surveys completed during class (Appendix 1). Surveys were identical, but the students were not made aware that either would be administered. The premodule asked students to relate either the topic “hormones” or “antibiotics” to the Five Core Concepts for Biological Literacy (1), which were provided to them on the survey. This premodule survey was completed upon the students arriving at lab in week one. The postmodule survey was administered in class immediately after experimental data collection and observations were completed. Students were provided with a copy of what they had written for the premodule survey and were asked to write down whether they could make new connections to the Five Core Concepts. After completion of the module, the instructor graded survey responses according to the rubric in Appendix 1 and, after removing student names from surveys, compiled common themes reported for each core concept. Data were also collected on whether student perspectives were broadened or not and whether or not each student identified more connections between the topics and core concepts. A quiz was administered to students after they read Davies (9) as homework to test their understanding of the article in general and the concept of hormesis in particular (Appendix 6). Each student’s grade for this course module was based on attendance, lab participation (successful completion of the labs and engagement in class discussions), the quiz on the article written by Davies (9), and performance on pre- and postmodule surveys (Appendix 7).

Sample data

All 17 groups of students in the three lab sections (69 students enrolled in spring 2014) observed Experiment 1 and inferred that hormone production by the fungus could be a possible explanation for increased fern growth, since a membrane physically separated the two organisms (Fig. 1). Students also determined that the membranes did not adversely impact fern growth (Fig. 1). Students were impressed that the fungus enhanced fern growth on all variations of BSM and that only slightly smaller plants were observed on 1/250 and 1/500 BSM. This illustrated to them that macronutrient concentrations commonly utilized in bioassays are unnecessarily high. It is important to note that students discussed in class that hormone production by the fungus was only one possible explanation for enhanced fern growth when the fungus was underneath the membrane and was a hypothesis requiring further investigation. It is possible that the fungus could be breaking down nutrients in the agar and facilitating fern growth, but the primary sources of nitrogen and phosphorus in the medium are already in an accessible form for the fern, so it is unlikely that the fern would rely heavily on the fungus for further nutrient breakdown before being able to absorb adequate amounts of nitrogen and phosphorus. Nonetheless, enhanced fern growth in the presence of the fungus, even when the two organisms were separated by a membrane, provides good evidence that this enhanced growth is somehow mitigated by the passage of chemicals between the two organisms.

FIGURE 1.

Ceratopteris richardii that germinated from spores on a) BSM agar medium, b) polycarbonate membrane on top of BSM agar medium, and c) polycarbonate membrane on top of fungi (Alternaria sp. BR) cultured on BSM agar medium. All images taken with a LeicaEZ4D at the same magnification, three weeks after sowing spores under constant light.

For Experiment 2, a few groups did not adequately spread plate and they could not collect reliable bacterial colony counts because excess liquid caused bacterial colonies to grow together. In laboratory section 2, students observed higher numbers of bacterial colonies at intermediate concentrations of the antibiotics tested (0.05 and 0.5 μg/mL) for four antibiotics; for two of the antibiotics, there were too few colonies to count and results were inconclusive (Fig. 2). Although results were not always consistent among laboratory sections, all three sections observed higher numbers of bacterial counts at intermediate concentrations of gentimycin and chloramphenicol. The variable results illustrated to students the need to repeat experiments with higher degrees of replication. Nonetheless, experiments did yield some supporting evidence for the hypothesis outlined in Davies (9) and were powerful in expanding student perspectives on the alternative roles antibiotic molecules might play in nature (see “evidence of student learning” in the Discussion section).

FIGURE 2.

Numbers of bacterial colonies per plate as a function of antibiotic type and concentration from laboratory section 2. Where bars are absent, the number of colonies was zero.

For Experiment 3, 13 of 17 student groups observed zones of clearing or slowed fungal growth around the test Streptomyces species, indicating that these isolates produce antifungal compounds; four groups did not observe any evidence of fungal growth inhibition (Fig. 3).

FIGURE 3.

Fungal inhibition assays on King’s B agar medium with novel bacterial isolates in the genus Streptomyces that were collected from outdoor air. After seven days of incubation at room temperature, bacteria in assays displayed in (a) and (b) did not inhibit fungal growth. Bacteria in assays displayed in (d), (e), and (f) inhibited fungal growth relative to the fungi only control (c).

Safety issues

All microbial cultures utilized in this laboratory module are biosafety level I organisms. Students are required to wear gloves when manipulating microorganisms and to follow proper aseptic technique. Bunsen burners and ethanol utilized for aseptic technique present fire hazards and require the instructor to properly demonstrate their use and to closely supervise student handling of these materials. Student identities were disassociated from pre- and postmodule surveys and quizzes prior to collecting the data presented in this manuscript. All data were collected in compliance with the Human Subjects Committee and Institutional Review Board at ISU.

DISCUSSION

Field testing

The module described within was administered to BIOL 2207 at ISU during three 165-minute class periods one week apart. The lecture section of this course meets for two 75-minute class periods per week. This class has a maximum of 72 students split into three laboratory sections. The laboratory section emphasizes experimental design, cellular structure and function, metabolism, and molecular biology.

The main objective of this module was accomplished. The students generated novel data that tested and provided supporting evidence for a hypothesis framed by Davies (9). Comments on postmodule surveys, which are discussed in more detail below, indicated that this experiment powerfully demonstrated to students that subinhibitory concentrations of antibiotics may enhance microbial growth and greatly expanded their knowledge on the topic of antibiotics. Many students also discussed the results of Experiment 1; they were amazed that fern growth was dramatically increased in the presence of the fungus and that ferns, even when grown alone, still grew well on medium containing 500-fold less macronutrients than commonly used bioassay conditions. After observing this, some students noted on the postmodule surveys that lab conditions can be pretty wasteful. They were able to liken this to potential overuse of fertilizer in agricultural settings after they went through an in-class exercise to calculate the concentrations of nutrients in the growth medium and compare them to those found in soils out in nature and seeing ferns grow perfectly well on medium with nutrient concentrations that mimicked nature. This was new to many students and one even noted that he or she had not really thought about nutrient concentrations found out in the natural environment. With respect to observing the dramatic impact that fungi had on fern growth and how that might translate to conducting applied science, one student noted that “knowing how organisms communicate could help and strengthen struggling ecosystems.” Experiment 3 provided primary data for a National Science Foundation Dimensions of Biodiversity project aimed at characterizing the biodiversity of the atmosphere. Seeing Streptomyces species isolated from the atmosphere inhibit fungal growth impacted student perspectives on how basic scientific research could fuel applied research, particularly for students who thought only biomedical companies produced antibiotics, as it showed them the value of microbial bioprospecting efforts. Postmodule surveys did not specifically poll student attitudes, but several students expressed positive feelings about the module: “I was excited to see the fungi vs. bacteria experiment. It got me thinking about the smaller things in life”; “I have a much greater appreciation of how living systems communicate.”

Evidence of student learning

Premodule surveys regarding the topics “antibiotics” and “hormones” were administered to 35 and 34 students, respectively, who were randomly distributed across all laboratory sections. On antibiotics, 97% of students surveyed reported new or broadened perspectives in the context of the Five Core Concepts for Biological Literacy (1). On hormones, the same was true for 83% of students surveyed. Module materials and experiments delved deeper into the topic of antibiotics, which may account for the differences in the percentage of students reporting new perspectives on the two topics.

On average, students saw connections between the topics and four of the Five Core Concepts in both pre- and postmodule surveys with no significant difference between the number of concepts referred to in pre- and postmodule surveys (paired t-test; p > 0.05). For each of the Five Core Concepts, McNemar’s tests demonstrated that students administered pre- and postmodule surveys on hormones were able to connect the topic of hormones with the concept of “Evolution” a significantly greater number of times on postmodule than on premodule surveys (p < 0.05), but there was no significant increase in the number of times other concepts were reported in postmodule surveys for either topic. However, the nature of the connections made between the topics and the core concepts differed substantially. Among 34 students surveyed on hormones, 20 new connections to core concepts were made on postmodule surveys; the 35 students surveyed on antibiotics made only nine such connections. Student perspectives on hormones reported on premodule surveys were much narrower in scope than those reported on antibiotics, which could have resulted from the recent or concurrent enrollment of many students in Human Anatomy and Physiology in which hormones are covered extensively.

The most commonly reported theme for each of the Five Core Concepts demonstrated expanded student perspectives on antibiotics (Table 3). The largest learning gains were observed for the “Systems” and “Pathways and Transformations of Energy and Matter” concepts (Table 1). For these two concepts, the most commonly reported themes on premodule surveys revolved around using antibiotics to treat infection, but on postmodule surveys the concept of hormesis and the role of “antibiotics” as chemical signaling molecules in nature were reported 29 and 30 times, respectively, on a total of 35 surveys (Table 3). Although students only scored an average of 78.5% on the quiz over Davies (9), the abundance of accurate descriptions of hormesis on postmodule surveys demonstrated that most students understood this concept by module’s end. In connection with “Information Flow, Exchange and Storage” and “Evolution” students described the role of “antibiotics” as cell signaling molecules and their possible role in the evolution of multicellularity (Table 3).

TABLE 3.

The most commonly reported theme, as inferred by the instructor, for each of the Five Core Concepts for Biological Literacy (Table 1; 1) on pre- and postmodule surveys.

Antibiotics (35 students surveyed)
Core Concept	Premodule surveys		Postmodule Surveys
	Theme reported	No. times reported	Theme reported	No. times reported
Evolution	Antibiotic resistance in microbes is increasing and medical advances are needed to combat this.	27	Instead of talking about antibiotic resistance evolving, we talked about low concentrations of antibiotics in nature that may have been responsible for the evolution of cooperative interactions.	6
Structure & Function	Understanding microbial and human body structure is necessary to understand how antibiotics work and how to design better ones.	19	Structure of bacterial cell walls, organelles and the structure of the antibiotic govern how it will kill pathogens.	14
Information Flow, Exchange and Storage	Antibiotics can change the behavior of microorganisms by inhibiting pathways for DNA replication, transcription, and translation, which are encoded in the genome.	7	At low concentrations, antibiotics may alter gene expression patterns and serve as signaling molecules (e.g. quorum sensing), allowing organisms to communicate	5
Pathways & Transformations of Energy & Matter	It is important to understand pathways involved in bacterial growth because this can help determine which antibiotics would be best at inhibiting growth.	8	Antibiotic effects on bacteria are concentration dependent (“hormesis”) and may positively affect growth as in Experiment 2.	29
Systems	Antibiotics need to be specific to attack pathogens while protecting the human system; humans experience negative side effects when normal/good microbial flora is harmed.	16	Antibiotics have more roles than humans think; out in nature antibiotics may serve as cell signaling molecules that allow microbes to communicate.	30
Hormones (34 students surveyed)
Core Concept	Premodule surveys		Postmodule surveys
	Theme reported	No. times reported	Theme reported	No. times reported
Evolution	Hormones have evolved over time through natural selection and regulate human sexual development and disease.	16	Hormones facilitate communication between different organisms (i.e. bacteria and plants) and have promoted cooperative interactions and symbioses to evolve over time.	16
Structure & Function	The 3D structure of hormones allows them to have specific functions in controlling human growth/physiology.	26	Hormone structure governs function (i.e. fungi may produce hormones that increase fern growth).	8
Information Flow, Exchange and Storage	Hormones connect pathways in the human body to control processes like growth, sexual development, sugar metabolism, muscle contraction, and neuron function.	21	Hormones are involved in information flow as signaling molecules that flow between organisms (Experiment 1) to promote growth or turn on defense responses.	11
Pathways & Transformations of Energy & Matter	Hormones regulate rates of chemical transformations in the human body to control production/utilization of energy, growth, sexual development and thyroid function.	14	Hormones regulate pathways for organismal growth, defense, and transformations of energy.	12
Systems	Hormones regulate how systems within the human body interact with each other/work together.	16	Plants and microbes produce hormones—not just humans and other animals—and can facilitate communication/growth.	22

The largest learning gain for the topic “hormones” was observed for the “Systems” concept (Table 3). Premodule surveys described hormones only as chemical messengers between systems within the human body, but postmodule surveys contained 22 reports of hormones as chemical signals passed between organisms like plants and microbes that can increase organismal growth in nature. Experiment 1 was powerful in expanding this perspective. For example postmodule commentary included, “Living systems are connected and interacting. This is something that I felt I knew but now I got to see how being interconnected in this case was beneficial for our fern.” Some students reported that they did not know that organisms like plants and bacteria could produce hormones. Other evidence for transformed thinking included, “before I was only thinking about hormonal function within one organism” and “…had not thought about hormones as something used to communicate between different organisms.”

Possible modifications

Some students struggled with spread plating bacteria onto agar plates. Incomplete spreading left pooled liquid on some plates, causing colonies to grow together and prevent accurate counts. Providing students with the opportunity to practice this technique before they work with actual samples might ameliorate this problem. Another modification may be to have the students plate different volumes (e.g. 50 and 100 μL) of two different dilutions to improve their chances of obtaining a countable number of colonies; this would also make it possible to test their pipetting accuracy by looking for the proper reduction in the number of colonies as dilution factors increased.

While this module was genuinely discovery-based and exposed students to the scientific process, the instructor closely guided the students because they had widely varied amounts of research experience. However, this module could be easily modified to provide advanced research experiences for majors in upper division courses. For instance, fern and fungal bioassays could be completed with a variety of different fungi from various culture collections and/or isolated from environmental samples collected by the students. Advanced exercises in statistical analysis could be completed by photographing ferns and quantifying their size using ImageJ, as described by Hill et al. (11). Macronutrient concentrations and stoichiometries could be altered to mimic those in a variety of natural environments or scenarios (e.g. anthropogenic nutrient loading).

Experiment 2 could be performed with different antibiotics across a wider range of concentrations and perhaps even under different nutrient conditions. Soil bacteria were used in the field test of this module, but different environmental samples (e.g. water) or even pure cultures could be utilized. To reduce the complexity of media preparation for Experiment 2, fewer antibiotics could be utilized.

Experiment 3 could be completed using a different array of bacterial isolates from public, private, or student-generated (depending on class time) culture collections. The bacterial cultures used in the field test of this module utilized Streptomyces species obtained from outdoor air (Weber et al., unpublished data), but this genus can be isolated from soils by using methods described by Kharel et al. (12).

This module discussed both hormones and antibiotics and their roles in plant and microbial communications in nature, but this module could be modified to include deeper discussion and research experience on only one of the topics.

Appendix 1: Pre- and postmodule surveys and suggested assessment rubrics

Appendix 2: Outline of interactive lecture/class discussion on hormones and preliminary experimental data

Appendix 3: Quantitative exercise to calculate molar concentration and stoichiometry of macronutrients in growth medium used by Contreras-Cornejo et al. (2009)

Appendix 4: Student and instructor protocols, growth medium recipes

Appendix 5: Outline of interactive lecture/class discussion on antibiotics and Streptomyces development

Appendix 6: Quiz over Davies (9)

Appendix 7: Suggested grading rubric for the module