a)  perform a Kirby-Bauer antimicrobial susceptibility test including application of antibiotic discs, measurement of zones of inhibition, and interpretation of the results.

b)  discuss the competing processes of antibiotic diffusion and microbial growth that lead to the formation of a zone of inhibition for a susceptible organism.

c)  describe the function of the Kirby-Bauer antibiotic susceptibility test.

d)  analyze and interpret the results for the class as compared to individual data.

e)  recognize that there is a wide range of variability within a bacterial species.

Students must have mastered the basics of aseptic technique before performing the Kirby-Bauer exercise.  Students also must be able to measure using the metric system and interpret data tables.

The strains utilized in field testing at Rogers State University included Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, and Staphylococcus aureus.  Of the six E. coli strains utilized, one is a lux HSL reporter strain, four are normally utilized in demonstrating lac operon action, and one is our common lab strain.  Three of the five S. marcescens strains (KWN, Nima, and db11) would be available from biological supply companies or from other various sources indicated in the literature.  Two of the S. marcescens strains were clinical in origin.  Three of the P. aeruginosa strains were clinical isolates, while four were isolates from various hot tubs, and the last was the commonly utilized strain PAO-1.  The three Staphylococcus strains actually included our lab strains of Staphylococcus aureus and S. epidermidis and one clinical strain of S. aureus that was antibiotic susceptible.

A variety of Escherichia coli strains may be purchased from the American Type Culture Collection.  Preceptrol strains are the most reasonably priced strains from the ATCC ($33 in 2007).  A current list of the preceptrol strains is available at: www.atcc.org/common/documents/pdf/preceptrol.pdf.  A number of the preceptrol strains are used in quality control for antimicrobial sensitivity testing.  To obtain more information about these strains, search the ATCC site (by ATCC number) for the individual strains; references are cited when known.   For example, the page for Escherichia coli ATCC 25922 gave a number of references, one of which was a full length Antimicrobial Agents and Chemotherapy paper (1) which utilized several of the preceptrol strains and presented zone of inhibition data. 

New England Biolabs will also provide cultures of various E. coli strains for the cost of shipping.  Many of these E. coli strains have inherent or plasmid-acquired antibiotic resistances.  Genotypes of these bacteria are supplied at the New England Biolabs website.  Different species and strains of bacteria can be purchased from other biological supply companies, including Wards and Carolina.  Selective media could also be used to isolate strains from various environments.  Additionally, many microbiologists may be able to provide strains from their strain collections.

 
B.  Growing the bacterial cultures
Prepare overnight (12 to 20 hour) broth cultures of the bacteria to be used in the exercise.  Cultures for most species can be grown up during the week before the experiment and refrigerated until needed; few of the bacteria will die in that short  period of time.  Inoculate broth tubes with bacteria from three to five well-isolated colonies of the same morphological type.  The tubes should contain 4 to 5 ml of sterile broth.  Several different broth media could be used to grow the bacteria for the exercise.  These include trypticase soy broth, nutrient broth, or Mueller-Hinton broth.  Several companies manufacture these media.  Powdered media or prepared media made by BD Diagnostic Systems can be purchased from a variety of scientific supply sources.  Cultures are grown without shaking at the appropriate temperature.  For example, S. aureus, E. coli, P. aeruginosa, and S. marcescens are all grown at 35 to 37°C.

 
C.  Adjust concentration of bacteria to provide standardized inocula
If the culture densities are not adjusted to the correct concentration, the exercise will still yield results, although the results will not be optimal.  If eyeballing concentrations is attempted, err on the side of lighter concentrations rather than higher concentrations.

• Visible light spectrophotometer and sterile cuvettes to measure A₆₂₅  or a McFarland standard of 0.5
• Sterile pipettes, 5-ml pipettes are adequate, approximately three per culture
• Sterile cuvettes, one per culture
• Pipette bulb
• Sterile 0.9% saline, approximately 10 ml per culture
• Discard container—a plastic bag, covered autoclaveable container, or similar container

 
Utilizing a visible light spectrophotometer to adjust bacterial concentration
After warming up a sufficient length of time and setting the wavelength for 625 nm, the spectrophotometer should be zeroed by setting the absorbance to "infinite" when nothing is present in the wavepath.  A blank should then be set up, consisting of a cuvette holding uninoculated bacterial broth, and the machine should be set to read an absorbance of zero.  Instructions for a commonly used spectrophotometer, the Spec 20, are available online at http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Growth_Curve/Spectrophotometer.htm.  Once the machine is set, the absorbance of a cuvette filled with bacteria can be read and adjusted utilizing the sterile saline.  A final absorbance between 0.08 and 0.10 is recommended.

 
Utilizing a McFarland standard to adjust bacterial concentration
The turbidity of the McFarland standard should be compared to the inoculum tube by holding them against a white card with contrasting black lines.  More details on procedures for using McFarland standards can be found in a variety of textbooks, e.g., Microbiology Laboratory Theory and Application (2), and web sources, such as the Centers for Disease Control and Prevention webpage www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera/ch9.pdf.  McFarland standards can be obtained from VWR; item 29447-318 costs approximately $8 each. 

The following recipe for preparing your own McFarland standard is as described in the package insert for the BBL antimicrobial susceptibility test discs. 

To prepare, add 0.5 ml of 0.048 M BaCl₂ (1.175% (wt/vol) BaCl₂ ^. 2H₂O) to 99.5 ml of 0.18 M [0.36 N] H₂SO₄ [1% (vol/vol)].  The turbidity should be verified by using a spectrophotometer with a 1 cm light path and matched cuvettes.  The absorbance at 625 nm should be 0.08 to 0.10.  This is roughly equivalent to 1 x 10⁸ to 2 x 10⁸ CFU/ml (CFU, colony forming units).

Preparation of materials 
1.  Petri plates filled with Mueller-Hinton agar
Mueller-Hinton is the standard agar for this test; if this media is not available nutrient agar or trypticase soy agar may be substituted, although the results will not be clinically relevant.  Mueller-Hinton agar, nutrient agar, and trypticase soy agar are manufactured by several companies, including BD Diagnostic Systems.  Powdered Mueller-Hinton agar or prepared plates may be purchased from many scientific suppliers. 

A wide variety of discs are available from many sources, including Carolina, Wards, VWR, and Fisher.  Discs should be stored in the refrigerator and kept with their desiccator packets.

• Lab report form, including data sheet for students to compile class data, and thought questions.  Students will discuss results with classmates at their lab station and respond to them in writing as their lab report.
• Ruler marked in mm, one per student or pair of students
• Interpretation table (available in lab manuals, package insert for antibiotic discs, and for selected antibiotics, as an appendix for this exercise); one per student or pair of students
• Blackboard and chalk to allow students to compile class data
• Appropriate discard location for contaminated materials

After lab is completed, a method is required for the appropriate disposal of contaminated materials.

Student Version.

References.

Suggestions for Determining Student Learning.

Student learning was assessed both formally and informally by examining student work. 

Informal assessment methods 

1.  Observe student inoculation techniques, results, and analysis.

2.  Listen to the students as they discuss the results.

The genesis of this project was a discussion with Dr. Kathryn Leyva, when we both taught at Midwestern University in Glendale, Arizona.  We did a trial run with undergraduate students in the degree completion program and presented this concept as a poster at the ASM Conference for Undergraduate Educators in 2001.

The exercise, as presented here, has been used with students twice at Rogers State University.  Both times it was used in Microbiology (BIOL 2124).  The only prerequisite for this course is the introductory biology course.  Microbiology is a prerequirement for students entering the A.S. nursing program at Rogers State University.  Other students may also take the course, especially those students desiring to obtain an A.S. in biology, as well as those students who will be transferring to other degree programs, such as optometry and pharmacy.  In the 2004 fall semester, it was used with two sections of Microbiology.  Eighteen of 40 students enrolled in the class agreed to participate in the field testing.  In the 2007 spring semester, 35 of 44 students agreed to participate in the field testing.

In fall 2005, we used several strains of Staphylococcus (two of these strains were really Staphylococcus epidermidis), Pseudomonas aeruginosa, Serratia marcescens, and Escherichia coli.  In 2007, only P.  aeruginosa, S. marcescens, and E. coli strains were used.

Both times, slightly more than 50% of the students presented conclusions which indicated that they realized that bacterial species could have variability and that testing of individual bacterial isolates would be essential to selecting the appropriate antibiotic for treatment.  Students received no information during the lab, in the handout, or during the lectures to indicate that different strains of bacteria might have different antimicrobial susceptibilities.  Those students who realized this was the case made the mental leap by examining their data and discussing what might cause these results.  Those students who did not reach these conclusions examined their lab procedures and results and reached conclusions that the different results were spurious.  No guiding questions were asked.

Feedback from other faculty at the ASM Conference for Undergraduate Educators indicated several reservations about using this approach in their classrooms.  Some indicated that they did not have a sufficient culture collection to support this exercise.  In the materials section, I have indicated several ways of resolving this issue.  These include obtaining strains of E. coli which have antibiotic resistance plasmids, isolating strains from the environment using standard approaches, and requesting attenuated or known strains of bacteria from culture collections, researchers, or other universities.  Other faculty liked this approach, but felt that they did not have the time to do it as a wet exercise.  In order to facilitate this, photographs of Kirby-Bauer plates from the 2007 exercise are being submitted.  This includes photographs of replicate plates for students to analyze how variable the results might be, as well as photographs of each of the strains used by the students.  Data from these exercises is also being submitted as appendices to this exercise.

Suggestions to Expand the Exercise.

This exercise could be expanded in several directions.

1.  Statistical analysis could be applied to the student data.

2.  Information regarding mechanisms of antibiotic resistance and transfer could be introduced.

3.  The Kirby-Bauer assay depends on standardization of several variables, including agar type, depth of agar in a plate, incubation temperature, and media pH.  Students could examine these variables, either during a discussion or in actual lab practice.

4.  A pre- and post-measure of student conceptions would be useful.

Consolidated Student Data

Representative Kirby-Bauer Plates

Zone Diameter Interpretive Chart for Selective Antibiotics

Review of Antibiotics and Their Range of Effectiveness

Illustrative Figures and Troubleshooting

Media Recipes