modified version of the Kirby-Bauer assay be-comes a vehicle for inquiry-based sciencewhen students choose what substances to testfor antimicrobial properties. This procedure

agar, forming a concentration gradient. Agar near thedisc has a higher concentration of antibiotic than theagar farther from the disc. If the organism is killed orits growth is inhibited, no bacteria will be found in theimmediate area around the disc. Using this modifica-tion of the assay, we encourage inquiry by providingstudents with plain paper discs, which they impreg-nate with the substance they choose for testing.

According to Lederman (2002), scientific inquirygoes beyond the development of process skills such asquestioning, observing, measuring, interpreting, andanalyzing data (inquiry as skills students learn) andjoins these processes with knowledge, reasoning, andcritical thinking to construct new scientific knowl-

Students learn aboutthe scientific process

and gain criticalthinking skills through

disc diffusion assays

Judith A . Scheppler , Nan Sethakorn , and Susan Styer

Acan be used for guided and authentic inquiry and pro-vides students opportunities to design experiments, draworiginal conclusions, and present their results.

The Kirby-Bauer assay, also called the disc diffu-sion assay, is a standard procedure used in clinicallaboratories to test the susceptibility of patients’ bacte-rial isolates to antibiotics (Bauer et al. 1966). In theassay, the bacteria are swabbed onto an agar plate, andpaper discs impregnated with antibiotics are placed onthe agar. The antibiotic diffuses from the disc into the

T h e S c i e n c e Te a c h e r5656565656

edge. Scientific inquiry, in short, canrefer to the systematic approaches usedby scientists in an effort to answerquest ions of interest (Chinn andH e m l o - S i l v e r 2 0 0 2 ; C h i n n a n dMalhotra 2002; Lederman 2002). Stu-dents should be able to ask scientificquestions and then design and conductinvestigations to produce data that willhelp them answer their questions. Asthey do this, students develop an un-derstanding of the scientific processand the tentative nature of scientificknowledge, as stated in the NationalScience Education Standards (NRC

Materials� bacterial cultures

� paper discs

� sterile swabs

� Petri dishes filled with nutrient agar

� liquid nutrient broth (premade medium is available)

� forceps

� test substances (brought by students)

� alcohol for sterilizing the forceps

� discard containers appropriate for bacteria

� bacterial incubator (optional)

Bacterial inoculation1. 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 swabagainst the inside of the tube above the fluid level. The swab is streaked in atleast three directions over the surface of the plate. Swab the surface of theagar heavily; do not leave any unswabbed agar areas. After completely swab-bing the plate, turn it 90° and repeat the swabbing process. (It is not neces-sary to remoisten the swab.) Run the swab around the circumference of theplate before discarding it in the discard bag.

3. Allow the surface to dry for about five minutes before placing discs onthe agar.

Disc preparation1. 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 ontothe side of the container, and then place the disc in the center of aquadrant of the plate. Press slightly on the disc with the forceps to secureit to the agar.

2. Repeat for the other quadrants with your other substances. Be sure to labelthe quadrant on the bottom of the plate with the test substance and label theplates with your name and date. (Don’t write all over the bottom where thediscs are because you will need to make measurements from the bottom.)

3. Incubate the Petri dishes overnight at 37oC; B. cereus cultures grow best at30oC. If no bacterial incubator is available, plates may be incubated atroom temperature for two to three days. Bacteria will grow more slowly atlower temperatures, but this will not affect the experimental results.

Bacterial disposalA standard and efficient method for killing bacteria in liquid cultures and oninoculated Petri dishes is by autoclaving them at 100oC and 15 psi for 20 minutes. Ifan autoclave is not available, then soaking the dishes in a sodium hypochloritesolution (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 finalvolume. After autoclaving or bleach treatment, the liquid may be disposed of downthe drain. Petri dishes may be discarded.

F I G U R E 1

Modified Kirby-Bauer disc diffusion assay.This experiment poses minimal risk to healthy individuals, but studentsshould use the standard safety precautions of wearing disposable glovesand goggles. Safer closed system plates are also available.

N ove m b e r 2 0 0 3 5757575757

T h e S c i e n c e Te a c h e r5858585858

(ATCC) serves as a good resource and provides safetyinformation. We used Escherichia coli (ATCC #9637),Staphylococcus epidermidis (ATCC #155), and Bacilluscereus (ATCC # 14579) because they are ones studentscan relate to. E. coli is found in the human intestine, S.epidermidis is found on the skin and mucus mem-branes, and B. cereus is a common environmental bac-terium that is also found in foods. These specificstrains are safe to handle, pose minimal risk to healthyindividuals, and require only standard and basic safetyprecautions.

Students bring test substances to class on the assignedday. Because IMSA is a residential school, many of thestudent 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 oftest substances (Figure 3), determine an appropriate con-trol, and include replicates. Different compounds may betested on the same bacterial strain, or one compound ondifferent strains. Students can vary the dilution of a testcompound, thus learning how to do serial dilutions. Theinstructor should review student experimental design be-fore students proceed with laboratory work.

To succeed, students must be careful handling Petridishes and equipment so that rogue microbes do notcontaminate the experiment. They should follow ster-

ile technique, minimize the time that plates areexposed to the air, and avoid touching the

agar surface with nonsterile objects.Assay interpretation is conducted by

measuring the zone of inhibitionaround each disc, which is the area

where no bacterial growth is occur-ring. With the plate upside down,students measure from one edge ofthe zone to the other edge (in mil-limeters) at its widest point. Byconvention, the disc diameter is in-cluded in that number. Students

average the replicates and calculatea standard deviation.

AssessmentThe outcome of this exercise was a stu-

dent-written laboratory report in the formatof a scientific paper (Booth 1994; Day 1998;McMillan 1988), including an introduction; amaterials 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 ofthe paper. In the introduction, students justified theirchoice of test materials and provided information aboutthe bacteria used. Students identified the active ingredi-

1996, p. 173–176; NRC 2000). This is also supportedby the Benchmarks for Science Literacy, which states thatstudents should understand the logic of an investiga-tion and be able to critically analyze the claims madefrom the data collected (AAAS 1993).

We have used this modified Kirby-Bauer assay(Figure 1, p. 57) as a classroom laboratory to helpintroduce students to inquiry and science content; asa guided inquiry activity (Martin-Hansen 2002), alsoknown as inquiry as pedagogy (Lederman 2002); andas an authentic inquiry (Chinn and Malhotra 2002),in which students work independently on a project.Chinn and Malhotra (2002) have described some ofthe features of authentic inquiry (Figure 2), and thisassay addresses many of these for both guided andauthentic inquiry.

Guided inquiryWhen the assay is used as a guided inquiry laboratory,students must provide an agent to test for antimicrobialproperties. The rest of the materials are supplied. Thiswas conducted in Scientific Inquiries (Torp et al. 1999),the core science class for sophomores at the Illinois Math-ematics and Science Academy (IMSA).

Teacher preparation includes making nutrient agarplates and inoculating and growing liquid cultures ofbacteria 18 to 24 hours before use. A variety of differ-ent bacteria may be used. The Ameri-can Type Culture Collection

We use bacteriastrains that are safe

to handle, poseminimal risk to

healthy individuals,and require only

standard and basicsafety precautions.

N ove m b e r 2 0 0 3 5959595959

ents in the test substances and ex-plained how the substance killed bac-teria or inhibited bacterial growth. Inthe discussion section, students ex-plained the results obtained.

Students were sometimes stumpedabout identifying the compoundsthat killed bacteria. For example,some mouthwashes list sodium fluo-ride as the active ingredient. Stu-dents erroneously thought that thiscompound killed bacteria, not notic-ing that the mouthwash madeanticavity claims. They needed tofully examine the ingredient list todetermine which compounds killbacteria because sodium fluoridedoes not . ( I t binds to teeth tostrengthen the enamel.)

Students had not previously writ-ten a report as a scientific paper re-quiring background research and thedrawing of conclusions based on evi-dence. Students did have experiencewriting about less open-ended classexperiments, where the results mainlyverified what was expected. Some re-sults were very confusing to students.For example, why did jelly inhibitbacterial growth even though it con-tained no preservatives? Why did stu-dent results show that Listerine, themost medicinal tasting mouthwash,was less effective in killing bacteriathan the less medicinal tasting Scope?Why might the three bacteria used inthis experiment not be completelykilled by mouthwashes? If soda pop isacidic, why didn’t it kill bacteria?What else is in soda?

These are the types of questions stu-dents had to answer in their discussionsection. To assist them, the teacher col-lected, reviewed, and returned labora-tory reports to students for revision.Many hints and suggestions were givenfor improvements. This process is notunlike writing a peer-reviewed paperfor publication, and it gives students aformative assessment before thesummative one.

Authentic inquiryScientists engage in authentic in-quiry when seeking an answer to a

F I G U R E 2

Features of authentic inquiry, from Chinn and Malhotra(2002), observed in guided inquiry and authentic inquiryusing the disc diffusion assay.

F I G U R E 3

Student-selected test substances and rationale for theirchoice, in the guided inquiry.

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

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 contactwith bacteria in food

Soda is acidic, thus a poor environment forliving organisms

Spicy foods may have antimicrobial properties

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T h e S c i e n c e Te a c h e r6060606060

question (Chinn and Malhotra 2002). At IMSA, thistype of project is conducted under the auspices of theStudent Inquiry Program (IMSA 2000). The purposeof this program is to give students guidance and struc-ture to design, develop, execute, troubleshoot, analyze,and communicate a project. The focusing question istheir own. Projects last one to two years.

Several students have chosen to use the disc diffu-sion assay to determine whether various food addi-tives and health products have antimicrobial proper-ties. The premise that spices in foods may preventbacterial growth is partly based on the knowledgethat before refrigeration was invented, spices wereadded to perishable goods to prevent spoilage. As acountry’s average temperature rises, the use of spiceswith antimicrobial properties also rises in meat-basedrecipes (Billing and Sherman 1998).

One former student examined a wide variety ofspices and health products over a two-year period onfour different bacteria (Figure 4). Test substance se-

lections were made based on thescientific literature as well as oncuriosity and published claimsmade by product manufacturers.Substances were tested a mini-mum of three times. Garlic wasthe most effective spice for inhib-iting bacterial growth.

A second student chose to focuson the antimicrobial properties ofchili peppers. It was determinedthat habanero hot sauce killed thefour test bacteria. Since capsaicin isthe molecule that makes the pep-pers hot (Cowan 1999), with theamount correlating with hotness,the student decided to test pure cap-saicin. It turned out that the capsai-cin only killed B. cereus. This resultled to discussion and considerationof why this might have occurred.

For both students, the inquiryproject involved much more thana simple one-time experiment.They prepared all solutions andbacterial media. They also had toinoculate bacteria into culture theday before setting up an experi-ment and mainta in bac ter ia lstocks. They set the project sched-ule and decided when to workand what experiments to do. Thestudents discussed results with theproject advisor, but this occurredless and less frequently as the stu-

F I G U R E 4

Student data on antimicrobial activity of variousspices and health products.

This table was presented as part of the poster presentation at the ASM annualmeeting. R = resistant—no zone of inhibition; I = intermediate—small zone ofinhibition; S = sensitive—large zone of inhibition; and NT = not tested.

Zones of inhibition of B. cereus tested with various spices.

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

N ove m b e r 2 0 0 3 6161616161

dents became more sophisticated in their scienceskills. They grappled with the difficulties of prepar-ing test substances. This included determining con-centration and solubility in solution and sterilizingthe solutions prior to use.

The bacteria used were chosen because they weresafe and readily available but were similar to thosethat might cause disease. B. cereus is a gram-positiverod-shaped bacterium. E. coli is a gram-negative rod-shaped bacterium. Staphylococcus capitis (ATCC#35661) is a gram-positive spherical bacterium. Sal-monella arizonae (ATCC #13314) is a gram-negativerod-shaped bacterium. The selection of these bacteriapromoted further discussion of the field of microbi-ology, delving into bacterial morphology, gram stain-ing, and even physiology. The availability of morescientific journals online makes it feasible for stu-dents to search and read the literature without goingto a un iver s i ty l ibrary . PubMed ( www.ncb i .nlm.nih.gov/PubMed) has a search engine for healthscience journals, providing abstracts and links tojournal websites.

Successful completion of an inquiry project re-quires communicating and defending the results. Eachspring at IMSA, inquiry students present their workorally to an audience that includes local scientists,businesspeople, and parents. Students also produceand display a poster as one might for a scientific con-ference, they are observed during their oral presenta-tions and posters are assessed. Students successfullyanswer many questions and address methodology,such as the solubility of test substances and its relationto obtaining an expected result. Each student alsowrites a paper in scientific format with references,which is also assessed in a manner similar to that de-scribed for the guided inquiry laboratory report.

This inquiry project provides the opportunity forone of the ultimate scientific experiences. One stu-dent presented her inquiry work at the American So-ciety for Microbiology (ASM) annual meeting in2001. The student interacted with educators, discuss-ing and defending her work for more than two hoursin a poster session.

The disc diffusion assay provides a mechanism forstudents to gain experience conducting scientific in-quiry. The two examples show how one might assiststudents in gaining skills and progressing from guidedto authentic inquiry. The assay also provides multipleopportunities for discussion of many areas of microbi-ology and cell biology, thus leveraging the laboratoryby including science content. n

Judith A. Scheppler (e-mail: quella@imsa.edu) is co-ordinator of student inquiry and director of theGrainger Center for Imagination and Inquiry; Nan

Sethakorn (e-mail: nsetha1@uic.edu), an IMSAgraduate, is a student at the University of Illinois atChicago; and Susan Styer (e-mail: sstyer@imsa.edu) is a science team member, all at Illinois Math-ematics and Science Academy, 1500 W. SullivanRoad, Aurora, IL 60506-1000.

Acknowledgments

We would like to thank Donald Dosch, Edwin Goebel, and StevenRogg for critical comments.

References

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