KIRBY-BAUER-DISK-DIFFUSION-SUSCEPTIBILITY-TEST.pptx

Kirby-Bauer Disk Diffusion
Susceptibility Test
Author: Multiple Authors
Citation: Multiple Authors. 2009. Kirby-bauer disk diffusion
susceptibility test.
Publication Date: December 2009

Kirby-Bauer disk diffusion susceptibility test
FIG. 1. Kirby-Bauer disk diffusion susceptibility test on coagulase-negative Staphylococcus aureus grown on Mueller-Hinton agar with tetracycline (30
µg), cephalothin (30 µg), erythromycin (15 µg), chloramphenicol (30 µg), vancomycin (30 µg), penicillin (10 µg), streptomycin (10 µg), and novobiocin
(30 µg). (Tasha L. Sturm, Cabrillo College, Aptos, CA)
American Society For Microbiology ©

FIG. 2. Kirby-Bauer disk diffusion susceptibility test on Staphylocuccus aureus. The image depicts measuring the zone of inhibition for tetracycline.
(Tasha L. Sturm, Cabrillo College, Aptos, CA)

FIG. 3. Kirby-Bauer disk diffusion susceptibility test on Pseudomonas aeruginosa. The results show sensitivity to amikacin and imipenem. Antibiotics
used include ampicillin (A), cefotaxime (Ce), co-trimoxazole (Co), ciprofloxacin (Cf), amoxicillin-clavulanic acid (Ac), ceftazidime (Ca), amikacin (Ak),
imipenem (I), and gentamicin (G). (Shashidhar Vishwanath, Kasturba Medical College, Karnataka, India)

FIG. 4. Kirby-Bauer disk diffusion test. A Mueller-Hinton agar plate was seeded with a lawn of Pseudomonas aeruginosa using a sterile cotton
swab. Antibiotic disks containing 30 µg of tetracycline (upper left), 30 µg of vancomycin (upper right), 10 µg of ampicillin (lower left), and 30 µg of
chloramphenicol (lower right) were dispensed on the agar surface, and the plate was incubated at 30°C overnight. The diameter of each zone was
measured in millimeters with a ruler and evaluated for susceptibility or resistance using the comparative standard method. (Anh-Hue Tu, Georgia
Southwestern State University, Americus)

FIG. 5. Kirby-Bauer disk diffusion test. A Mueller-Hinton agar plate was seeded with a lawn of Pseudomonas aeruginosa (top half) and Serratia
marcescens (bottom half) using sterile cotton swabs. For plate A, antibiotic disks containing 30 µg of chloramphenicol (top and bottom left), 15 µg of
erythromycin (top and bottom middle), and 30 µg of ampicillin (top and bottom right) were dispensed on the agar surface. For plate B, antibiotic disks
containing 25 µg of sulfisoxazole (top and bottom left) and 30 µg of ceftriaxone (top and bottom right) were dispensed on the agar surface. Both
plates were incubated at 30°C overnight and the diameter of each zone was measured in millimeters and evaluated for susceptibility or resistance
using the comparative standard method. (Anh-Hue Tu, Georgia Southwestern State University, Americus)

FIG. 6. Kirby-Bauer disk diffusion test. Mueller-Hinton agar plates were seeded with Pseudomonas aeruginosa, Serratia marcescens, and
Staphylococcus aureus. Four antibiotic disks were dispensed on each plate. The disks contained 10 µg of ampicillin (top left), 30 µg of tetracycline
(top right), 30 µg of chloramphenicol (bottom left), and 30 µg of vancomycin (bottom right). All three plates were incubated at 30°C overnight. The
diameter of each zone was measured in millimeters and evaluated for resistance or susceptibility using the comparative standard method. Antibiotic
susceptibility was compared between the three strains of bacteria. (Anh-Hue Tu, Georgia Southwestern State University, Americus)

FIG. 7. Kirby-Bauer disk diffusion test. Mueller-Hinton agar plates were seeded with Pseudomonas aeruginosa and Staphylococcus aureus. Four
antibiotic disks were dispensed on each plate. The disks contained 30 µg of tetracycline (top left), 30 µg of vancomycin (top right), 10 µg of ampicillin
(bottom left), and 30 µg of chloramphenicol (bottom right). Both plates were incubated at 30°C overnight. The diameter of each zone was measured
in millimeters and evaluated for susceptibility or resistance using the comparative standard method. Antibiotic susceptibility was compared between
the two strains of bacteria. (Anh-Hue Tu, Georgia Southwestern State University, Americus)

FIG. 8. Kirby-Bauer disk diffusion test. Mueller-Hinton agar plates were seeded with Pseudomonas aeruginosa, Serratia marcescens, and
Staphylococcus aureus. Four antibiotic disks were dispensed on each plate. The disks contained 30 µg of chloramphenicol (top left), 10 µg of
ampicillin (top right), 30 µg of vancomycin (bottom left), and 30 µg of tetracycline (bottom right). Both plates were incubated at 30°C overnight. The
diameter of each zone was measured in millimeters and evaluated for resistance or susceptibility using the comparative standard method. Antibiotic
susceptibility was compared between the three strains of bacteria. (Anh-Hue Tu, Georgia Southwestern State University, Americus, GA)

FIG. 9. Kirby-Bauer disk diffusion test. A Mueller-Hinton agar plate was seeded with a lawn of Staphylococcus aureus using a sterile cotton
swab. Antibiotic disks containing 30 µg of ceftriaxone (upper left), 25 µg of sulfisoxazole (upper right), and 30 µg of polymixin B (lower right) were
dispensed on the agar surface. The plate was incubated at 30°C overnight. The diameter of each zone was measured in millimeters with a ruler and
evaluated for resistance or susceptibility using the comparative standard method. (Anh-Hue Tu, Georgia Southwestern State University, Americus, GA)

FIG. 10. Kirby-Bauer disk diffusion test on Escherichia coli grown on Mueller-Hinton agar using antibiotic disks containing 10 µg of ampicillin (AM 10),
30 µg of tetracycline (Te 30), and 10 IU of penicillin (P 10) after a 24-hour incubation. (Jackie Peltier Horn, Houston Baptist University, Houston, TX)

FIG. 11. Kirby-Bauer disk diffusion test. Oxacillin, doxycycline, and cefoxitin antibiotic disks were placed on Mueller-Hinton agar after plating with
Staphylococcus aureus. The plate is shown just prior to incubation. (Clarissa L. Kaup, Bellevue University, Bellevue, NE; J.L. Henriksen, Bellevue
University, Bellevue, NE)

FIG. 12. Kirby-Bauer disk diffusion test on Staphylococcus aureus grown on Mueller-Hinton agar. Zones of sensitivity are shown for oxacillin (15 mm),
cefoxitin (30 mm), and doxycycline (27 mm) after 48 hours of incubation at 37°C. (Clarissa L. Kaup, Bellevue University, Bellevue, NE; J.L. Henriksen,
Bellevue University, Bellevue, NE)

FIG. 13. Kirby-Bauer disk diffusion test on Staphylococcus aureus grown on Mueller-Hinton agar. Staphylococcus aureus shows sensitivity to oxacillin
(15-mm zone) after 48 hours of incubation at 37°C. (Clarissa L. Kaup, Bellevue University, Bellevue, NE; J.L. Henriksen, Bellevue University, Bellevue,
NE)

FIG. 14. Kirby-Bauer disk diffusion test on Staphylococcus aureus grown on Mueller-Hinton agar. Staphylococcus aureus shows decreased sensitivity
to cefoxitin (11-mm zone) and doxycycline (26-mm zone) and resistance to oxacillin after 48 hours of incubation at 37°C. (Clarissa L. Kaup, Bellevue
University, Bellevue, NE; J.L. Henriksen, Bellevue University, Bellevue, NE)

FIG. 15. Kirby-Bauer disk diffusion test on Staphylococcus aureus grown on Mueller-Hinton agar. Staphylococcus aureus shows resistance to oxacillin
after 48 hours of incubation at 37°C. (Clarissa L. Kaup, Bellevue University, Bellevue, NE, J.L. Henriksen, Bellevue University, Bellevue, NE)

McFarland standards
FIG. 16. McFarland standards (left to right) 0.5, 1.0, 2.0, 3.0, positioned in front of a Wickerham card. McFarland standards are used to prepare
bacterial suspensions to a specified turbidity. In the Kirby-Bauer disk diffusion susceptibility test protocol, the bacterial suspension of the organism to
be tested should be equivalent to the 0.5 McFarland standard. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Inoculation of the test plate
FIG. 17. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the test plate. Step 2. Rotate the swab against the side of the tube while
applying pressure to remove excess liquid from the swab prior to inoculating the plate. (Jan Hudzicki, University of Kansas Medical Center, Kansas City,
KS)

Inoculation of the Mueller-Hinton agar plate
FIG. 18. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton agar plate. Step 3. Inoculate the plate with the test
organism by streaking the swab in a back-and-forth motion very close together as you move across and down the plate. Rotate the plate 60° and
repeat this action. Rotate the plate once more and repeat the streaking action. This method ensures an even distribution of inoculum that will result
in a confluent lawn of growth. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Diagram illustrating the pattern the swab should follow as it is drawn across the plate
FIG. 19. Inoculation of the Mueller-Hinton agar plate, diagram illustrating the pattern the swab should follow as it is drawn across the plate. (Jan
Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Inoculation of the Mueller-Hinton agar plate
FIG. 20. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton agar plate. Step 4. After streaking the Mueller-
Hinton agar plate as described in Step 3, rim the plate with the swab by running the swab around the edge of the entire plate to pick up any excessive
inoculum that may have been splashed near the edge. The arrow indicates the path of the swab. (Jan Hudzicki, University of Kansas Medical Center,
Kansas City, KS) American Society For Microbiology ©

Set the dispenser over the plate
FIG. 21. Kirby-Bauer disk diffusion susceptibility test protocol, placement of antimicrobial disks using an automated disk dispenser. Step 1. An
automatic disk dispenser can be used to place multiple disks simultaneously on a Mueller-Hinton agar plate. Set the dispenser over the plate. (Jan

Place the palm of your hand on the top of the handle
FIG. 22. Place the palm of your hand on the top of the handle. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Press down firmly and completely to dispense the disks
FIG. 23. Press down firmly and completely to dispense the disks. The spring-loaded handle will return to the original position when pressure is
removed. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Place the Mueller-Hinton agar plate over the disk template
FIG. 24. Kirby-Bauer disk diffusion susceptibility test protocol, placement of antimicrobial disks using forceps to manually place the disks. Step
1. Antimicrobial disks can be manually placed on the Mueller-Hinton agar plate if desired. Place the Mueller-Hinton agar plate over the disk
template. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Remove one disk from the cartridge using forceps that have been sterilized
FIG. 25. Remove one disk from the cartridge using forceps that have been sterilized. (Jan Hudzicki, University of Kansas Medical Center, Kansas City,
KS)

Lift the lid of the plate and place the disk over one of the positioning marks
FIG. 26. Lift the lid of the plate and place the disk over one of the positioning marks. (Jan Hudzicki, University of Kansas Medical Center, Kansas City,
KS)

Press the disk with the forceps to ensure complete contact with the agar surface. Replace the lid of the plate between disks to minimize exposure to
airborne contaminants.
FIG. 27. Press the disk with the forceps to ensure complete contact with the agar surface. Replace the lid of the plate between disks to minimize
exposure to air-borne contaminants. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Manual disk placement template for 8 disks on a 100-mm plate
FIG. 28. Manual disk placement template for eight disks on a 100-mm plate. Place the Mueller-Hinton agar plate on the figure so that the edge of the
plate lines up with the outer circle. Remove the lid from the plate and place one antimicrobial disk over each dark gray circle. If fewer than eight
antimicrobial disks are used, adjustments can be made to the spacing of the disks. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Measuring zones of inhibition: gray shading represents a confluent lawn of bacterial growth
FIG. 29. Measuring zones of inhibition. Gray shading represents a confluent lawn of bacterial growth. The white circle represents no growth of the
test organism. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Using a ruler or caliper measure each zone with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black,
non-reflecting surface illuminated with reflected light.
FIG. 30. Kirby-Bauer disk diffusion susceptibility test protocol, measuring zone sizes. Using a ruler or caliper measure each zone with the unaided eye
while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light. (Jan

The size of the zone for this organism-antimicrobial combination is 26 mm
FIG. 31. The size of the zone for this organism-antimicrobial combination is 26 mm. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Alternate method for measuring zones
FIG. 32. Kirby-Bauer disk diffusion susceptibility test protocol, an alternate method for measuring zones. If the zones of adjacent antimicrobial disks
overlap, the zone diameter can be determined by measuring the radius of the zone. Measure from the center of the antimicrobial disk to a point on
the circumference of the zone where a distinct edge is present. Multiply this measurement by 2 to determine the diameter of the zone of
inhibition. In this example, the radius of the zone is 16 mm. Multiply this measurement by 2 to determine a zone size of 32 mm for this organism-
antimicrobial combination. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)

Staphylococcus saprophyticus resistant to novobiocin
FIG 33. Staphylococcus saprophyticus was diluted in TSB (.25 mls 24 hr old broth culture into 5mls TSB) then swabbed onto a Mueller Hinton agar for
confluent growth a Novobiocin disc (30 µg) then incubated at 37oC for 48 hrs. S. saprophyticus is resistant (no zone of inhibition) to novobiocin. This
test is used as a way to differentiate between Staphylococcus saprophyticus (resistant), Staphylococcus aureus and Staphylococcus epidermidis (both
sensitive). Staphylococcus saprophyticus is ~9mm. (Tasha Sturm, Cabrillo College, Aptos, CA)

Staphylococcus epidermidis is sensitive with a zone of inhibition
FIG. 34. Staphylococcus epidermidis was diluted in TSB (.25 mls 24 hr old broth culture into 5mls TSB) then swabbed onto a Mueller Hinton agar for
confluent growth a Novobiocin disc (30 µg) then incubated at 37oC for 48 hrs. Staphylococcus epidermidis is sensitive with a zone of inhibition
>20mm. This test is used as a way to differentiate between Staphylococcus saprophyticus (resistant), Staphylococcus aureus and Staphylococcus
epidermidis (both sensitive). Staphylococcus epidermidis is ~37mm. (Tasha Sturm, Cabrillo College, Aptos, CA)

Staphylococcus aureus is sensitive with a zone of inhibition
FIG. 35. Staphylococcus aureus was diluted in TSB (.25 mls 24 hr old broth culture into 5mls TSB) then swabbed onto a Mueller Hinton agar for
confluent growth a Novobiocin disc (30 µg) then incubated at 37oC for 48 hrs. Staphylococcus aureus is sensitive with a zone of inhibition
>20mm. This test is used as a way to differentiate between Staphylococcus saprophyticus (resistant), Staphylococcus aureus and Staphylococcus
epidermidis (both sensitive). Staphylococcus aureus is ~41mm. (Tasha Sturm, Cabrillo College, Aptos, CA)

Bacitracin test
FIG. 36. Bacitracin test done on a lawn of Streptococcus pyogenes grown on blood agar. The zone of inhibition around the bacitracin disc,
approximately 14mm measuring the entire width of the zone, indicates sensitivity. The zone of inhibition is red because the red blood cells did not
lyse. Grown for 24 hrs at 370 C. (Tasha Sturm, Cabrillo College, Aptos, CA)

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