Students learn about the scientific process and gain critical thinking skills through disc diffusion assays

Students learn about the scientific process and gain critical thinking skills through disc diffusion assays Judith A. Scheppler, Nan Sethakorn, and Susan Styer A modified version of the Kirby-Bauer assay becomes a vehicle for inquiry-based science when students choose what substances to test for antimicrobial properties. This procedure can be used for guided and authentic inquiry and provides students opportunities to design experiments, draw original conclusions, and present their results. The Kirby-Bauer assay, also called the disc diffusion assay, is a standard procedure used in clinical laboratories to test the susceptibility of patients bacterial isolates to antibiotics (Bauer et al. 1966). In the assay, the bacteria are swabbed onto an agar plate, and paper discs impregnated with antibiotics are placed on the agar. The antibiotic diffuses from the disc into the 56 T h e S c i e n c e Te a c h e r agar, forming a concentration gradient. Agar near the disc has a higher concentration of antibiotic than the agar farther from the disc. If the organism is killed or its growth is inhibited, no bacteria will be found in the immediate area around the disc. Using this modification of the assay, we encourage inquiry by providing students with plain paper discs, which they impregnate with the substance they choose for testing. According to Lederman (2002), scientific inquiry goes beyond the development of process skills such as questioning, observing, measuring, interpreting, and analyzing data (inquiry as skills students learn) and joins these processes with knowledge, reasoning, and critical thinking to construct new scientific knowl-

FIGURE 1 Modified Kirby-Bauer disc diffusion assay. Materials bacterial cultures paper discs sterile swabs Petri dishes filled with nutrient agar liquid nutrient broth (premade medium is available) forceps This experiment poses minimal risk to healthy individuals, but students should use the standard safety precautions of wearing disposable gloves and goggles. Safer closed system plates are also available. test substances (brought by students) alcohol for sterilizing the forceps discard containers appropriate for bacteria bacterial incubator (optional) Bacterial inoculation 1. Divide each nutrient agar plate into four separate quadrants, labeling the bottom of the plate, not the lid. 2. Swab the plates with the bacteria. To do this, dip a sterile swab into the bacterial suspension broth and remove the excess fluid by pressing the swab against the inside of the tube above the fluid level. The swab is streaked in at least three directions over the surface of the plate. Swab the surface of the agar heavily; do not leave any unswabbed agar areas. After completely swabbing the plate, turn it 90 and repeat the swabbing process. (It is not necessary to remoisten the swab.) Run the swab around the circumference of the plate before discarding it in the discard bag. 3. Allow the surface to dry for about five minutes before placing discs on the agar. edge. Scientific inquiry, in short, can refer to the systematic approaches used by scientists in an effort to answer questions of interest (Chinn and Hemlo-Silver 2002; Chinn and Malhotra 2002; Lederman 2002). Students should be able to ask scientific questions and then design and conduct investigations to produce data that will help them answer their questions. As they do this, students develop an understanding of the scientific process and the tentative nature of scientific knowledge, as stated in the National Science Education Standards (NRC Disc preparation 1. Sterilize the forceps by dipping them in alcohol, then use them to pick up a sterile disc. Dip the disc into the test substance, blot excess liquid onto the side of the container, and then place the disc in the center of a quadrant of the plate. Press slightly on the disc with the forceps to secure it to the agar. 2. Repeat for the other quadrants with your other substances. Be sure to label the quadrant on the bottom of the plate with the test substance and label the plates with your name and date. (Don t write all over the bottom where the discs are because you will need to make measurements from the bottom.) 3. Incubate the Petri dishes overnight at 37 o C; B. cereus cultures grow best at 30 o C. If no bacterial incubator is available, plates may be incubated at room temperature for two to three days. Bacteria will grow more slowly at lower temperatures, but this will not affect the experimental results. Bacterial disposal A standard and efficient method for killing bacteria in liquid cultures and on inoculated Petri dishes is by autoclaving them at 100 o C and 15 psi for 20 minutes. If an autoclave is not available, then soaking the dishes in a sodium hypochlorite solution (10 percent common household bleach) for at least one hour works well. For liquid cultures, add bleach so that the bleach is about 10 percent of the final volume. After autoclaving or bleach treatment, the liquid may be disposed of down the drain. Petri dishes may be discarded. November 2003 57

1996, p. 173 176; NRC 2000). This is also supported by the Benchmarks for Science Literacy, which states that students should understand the logic of an investigation and be able to critically analyze the claims made from the data collected (AAAS 1993). We have used this modified Kirby-Bauer assay (Figure 1, p. 57) as a classroom laboratory to help introduce students to inquiry and science content; as a guided inquiry activity (Martin-Hansen 2002), also known as inquiry as pedagogy (Lederman 2002); and as an authentic inquiry (Chinn and Malhotra 2002), in which students work independently on a project. Chinn and Malhotra (2002) have described some of the features of authentic inquiry (Figure 2), and this assay addresses many of these for both guided and authentic inquiry. Guided inquiry When the assay is used as a guided inquiry laboratory, students must provide an agent to test for antimicrobial properties. The rest of the materials are supplied. This was conducted in Scientific Inquiries (Torp et al. 1999), the core science class for sophomores at the Illinois Mathematics and Science Academy (IMSA). Teacher preparation includes making nutrient agar plates and inoculating and growing liquid cultures of bacteria 18 to 24 hours before use. A variety of different bacteria may be used. The American Type Culture Collection We use bacteria strains that are safe to handle, pose minimal risk to healthy individuals, and require only standard and basic safety precautions. (ATCC) serves as a good resource and provides safety information. We used Escherichia coli (ATCC #9637), Staphylococcus epidermidis (ATCC #155), and Bacillus cereus (ATCC # 14579) because they are ones students can relate to. E. coli is found in the human intestine, S. epidermidis is found on the skin and mucus membranes, and B. cereus is a common environmental bacterium that is also found in foods. These specific strains are safe to handle, pose minimal risk to healthy individuals, and require only standard and basic safety precautions. Students bring test substances to class on the assigned day. Because IMSA is a residential school, many of the student test compounds are chosen based on convenience. Students bring what they can obtain from the cafeteria, what they have in the dormitories, and so forth. Students should provide rationales for their choice of test substances (Figure 3), determine an appropriate control, and include replicates. Different compounds may be tested on the same bacterial strain, or one compound on different strains. Students can vary the dilution of a test compound, thus learning how to do serial dilutions. The instructor should review student experimental design before students proceed with laboratory work. To succeed, students must be careful handling Petri dishes and equipment so that rogue microbes do not contaminate the experiment. They should follow sterile technique, minimize the time that plates are exposed to the air, and avoid touching the agar surface with nonsterile objects. Assay interpretation is conducted by measuring the zone of inhibition around each disc, which is the area where no bacterial growth is occurring. With the plate upside down, students measure from one edge of the zone to the other edge (in millimeters) at its widest point. By convention, the disc diameter is included in that number. Students average the replicates and calculate a standard deviation. Assessment The outcome of this exercise was a student-written laboratory report in the format of a scientific paper (Booth 1994; Day 1998; McMillan 1988), including an introduction; a materials and methods section in a past-tense narrative style; a results section, including tables, if appropriate; a discussion and conclusion; and a reference list, with embedded citations in the body of the paper. In the introduction, students justified their choice of test materials and provided information about the bacteria used. Students identified the active ingredi- 58 The Science Teacher

ents in the test substances and explained how the substance killed bacteria or inhibited bacterial growth. In the discussion section, students explained the results obtained. Students were sometimes stumped about identifying the compounds that killed bacteria. For example, some mouthwashes list sodium fluoride as the active ingredient. Students erroneously thought that this compound killed bacteria, not noticing that the mouthwash made anticavity claims. They needed to fully examine the ingredient list to determine which compounds kill bacteria because sodium fluoride does not. (It binds to teeth to strengthen the enamel.) Students had not previously written a report as a scientific paper requiring background research and the drawing of conclusions based on evidence. Students did have experience writing about less open-ended class experiments, where the results mainly verified what was expected. Some results were very confusing to students. For example, why did jelly inhibit bacterial growth even though it contained no preservatives? Why did student results show that Listerine, the most medicinal tasting mouthwash, was less effective in killing bacteria than the less medicinal tasting Scope? Why might the three bacteria used in this experiment not be completely killed by mouthwashes? If soda pop is acidic, why didn t it kill bacteria? What else is in soda? These are the types of questions students had to answer in their discussion section. To assist them, the teacher collected, reviewed, and returned laboratory reports to students for revision. Many hints and suggestions were given for improvements. This process is not unlike writing a peer-reviewed paper for publication, and it gives students a formative assessment before the summative one. Authentic inquiry Scientists engage in authentic inquiry when seeking an answer to a FIGURE 2 Features of authentic inquiry, from Chinn and Malhotra (2002), observed in guided inquiry and authentic inquiry using the disc diffusion assay. Characteristics Guided inquiry Authentic inquiry Generating research questions No Yes Selecting variables No Yes Developing simple controls Yes Yes Developing relatively complex controls No Yes Making multiple observations No Yes Observing intervening variables No No Using analog models Yes Yes Simple transformation of observations Yes Yes Complex transformation of observations No Yes Consideration of methodological flaws Yes Yes Developing theories and mechanisms Yes Yes Multiple studies of the same type No Yes Multiple studies of different types No Yes Studying expert research reports Yes Yes FIGURE 3 Student-selected test substances and rationale for their choice, in the guided inquiry. Test substance Mouthwashes, toothpastes, laundry detergents, hand sanitizer Chicken soup Contact lens cleaner Cream cheese Jelly Dishwashing detergent Soda Hot sauce Rationale Advertised to kill bacteria Good for sick people Cleans lenses, perhaps by killing bacteria Contains preservatives to keep it fresh Contains preservative (although it does not, student thought it did) Cleans dishes that may have come in contact with bacteria in food Soda is acidic, thus a poor environment for living organisms Spicy foods may have antimicrobial properties November 2003 59

Zones of inhibition of B. cereus tested with various spices. FIGURE 4 Student data on antimicrobial activity of various spices and health products. This table was presented as part of the poster presentation at the ASM annual meeting. R = resistant no zone of inhibition; I = intermediate small zone of inhibition; S = sensitive large zone of inhibition; and NT = not tested. Bacteria E. coli S. arizonae S. capitis B. cereus Salt I R R R Pepper R R R R Rosemary R R R R Lemon R R R R Lime R R R R Garlic S I S S Cloves I I I I Curry powder R R R I Cinnamon R NT NT NT Nutmeg R NT NT NT Paprika R NT NT NT Tumeric R NT NT NT Tea tree oil S I I I Sun breeze oil I S I I Chili pepper R R R R Oregano R NT NT NT question (Chinn and Malhotra 2002). At IMSA, this type of project is conducted under the auspices of the Student Inquiry Program (IMSA 2000). The purpose of this program is to give students guidance and structure to design, develop, execute, troubleshoot, analyze, and communicate a project. The focusing question is their own. Projects last one to two years. Several students have chosen to use the disc diffusion assay to determine whether various food additives and health products have antimicrobial properties. The premise that spices in foods may prevent bacterial growth is partly based on the knowledge that before refrigeration was invented, spices were added to perishable goods to prevent spoilage. As a country s average temperature rises, the use of spices with antimicrobial properties also rises in meat-based recipes (Billing and Sherman 1998). One former student examined a wide variety of spices and health products over a two-year period on four different bacteria (Figure 4). Test substance selections were made based on the scientific literature as well as on curiosity and published claims made by product manufacturers. Substances were tested a minimum of three times. Garlic was the most effective spice for inhibiting bacterial growth. A second student chose to focus on the antimicrobial properties of chili peppers. It was determined that habanero hot sauce killed the four test bacteria. Since capsaicin is the molecule that makes the peppers hot (Cowan 1999), with the amount correlating with hotness, the student decided to test pure capsaicin. It turned out that the capsaicin only killed B. cereus. This result led to discussion and consideration of why this might have occurred. For both students, the inquiry project involved much more than a simple one-time experiment. They prepared all solutions and bacterial media. They also had to inoculate bacteria into culture the day before setting up an experiment and maintain bacterial stocks. They set the project schedule and decided when to work and what experiments to do. The students discussed results with the project advisor, but this occurred less and less frequently as the stu- 60 The Science Teacher

dents became more sophisticated in their science skills. They grappled with the difficulties of preparing test substances. This included determining concentration and solubility in solution and sterilizing the solutions prior to use. The bacteria used were chosen because they were safe and readily available but were similar to those that might cause disease. B. cereus is a gram-positive rod-shaped bacterium. E. coli is a gram-negative rodshaped bacterium. Staphylococcus capitis (ATCC #35661) is a gram-positive spherical bacterium. Salmonella arizonae (ATCC #13314) is a gram-negative rod-shaped bacterium. The selection of these bacteria promoted further discussion of the field of microbiology, delving into bacterial morphology, gram staining, and even physiology. The availability of more scientific journals online makes it feasible for students to search and read the literature without going to a university library. PubMed (www.ncbi. nlm.nih.gov/pubmed) has a search engine for health science journals, providing abstracts and links to journal websites. Successful completion of an inquiry project requires communicating and defending the results. Each spring at IMSA, inquiry students present their work orally to an audience that includes local scientists, businesspeople, and parents. Students also produce and display a poster as one might for a scientific conference, they are observed during their oral presentations and posters are assessed. Students successfully answer many questions and address methodology, such as the solubility of test substances and its relation to obtaining an expected result. Each student also writes a paper in scientific format with references, which is also assessed in a manner similar to that described for the guided inquiry laboratory report. This inquiry project provides the opportunity for one of the ultimate scientific experiences. One student presented her inquiry work at the American Society for Microbiology (ASM) annual meeting in 2001. The student interacted with educators, discussing and defending her work for more than two hours in a poster session. The disc diffusion assay provides a mechanism for students to gain experience conducting scientific inquiry. The two examples show how one might assist students in gaining skills and progressing from guided to authentic inquiry. The assay also provides multiple opportunities for discussion of many areas of microbiology and cell biology, thus leveraging the laboratory by including science content. n Judith A. Scheppler (e-mail: quella@imsa.edu) is coordinator of student inquiry and director of the Grainger Center for Imagination and Inquiry; Nan Sethakorn (e-mail: nsetha1@uic.edu), an IMSA graduate, is a student at the University of Illinois at Chicago; and Susan Styer (e-mail: sstyer@imsa. edu) is a science team member, all at Illinois Mathematics and Science Academy, 1500 W. Sullivan Road, Aurora, IL 60506-1000. Acknowledgments We would like to thank Donald Dosch, Edwin Goebel, and Steven Rogg for critical comments. References American Association for the Advancement of Science (AAAS). 1993. Benchmarks for Science Literacy: Project 2061. New York: Oxford University Press. Bauer, A.W., W.M.M. Kirby, J.C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disc method. Technical Bulletin of the Registry of Medical Technologists 36(3): 49 52. Billing, J., and P.W. Sherman. 1998. Antimicrobial functions of spices: Why some like it hot. The Quarterly Review of Biology 73(1): 3 49. Booth, V. 1994. Communicating in Science. 2nd ed. New York: Cambridge University Press. Chinn, C.A., and C.E. Hemlo-Silver. 2002. Authentic inquiry: Introduction to the special section. Science Education 86(2): 171 174. Chinn, C.A., and B.A. Malhotra. 2002. Epistemologically authentic inquiry in schools: A theoretical framework for evaluating inquiry tasks. Science Education 86(2): 175 218. Cowan, M. 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12(4): 564 682. Day, R.A. 1998. How to Write and Publish a Scientific Paper. 5th ed. Phoenix, Ariz.: Oryx Press. Illinois Mathematics and Science Academy (IMSA). 2000. Student Inquiry and Research Program. Aurora, Ill.: Illinois Mathematics and Science Academy. Lederman, N.G. 2002. Scientific inquiry and nature of science as a meaningful context for learning in science. In Science Literacy for the Twenty-First Century. Edited by S.P. Marshall, J.A. Scheppler, M.J. Palmisano. Amherst, N.Y.: Prometheus Books. Martin-Hansen, L. 2002. Defining inquiry: Exploring the many types of inquiry in the science classroom. The Science Teacher 69(2): 34 37. McMillan, V.E. 1988. Writing Papers in the Biological Sciences. New York: St. Martin s Press. National Research Council (NRC). 1996. National Science Education Standards. Washington, D.C.: National Academy Press. National Research Council (NRC). 2000. Inquiry and the National Science Education Standards. Washington, D.C.: National Academy Press. Torp, L., D. Dosch, D. Hinterlong, and S. Styer. 1999. Scientific Inquiries: A New Beginning for Science at IMSA. Aurora, Ill.: Illinois Mathematics and Science Academy. November 2003 61