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Kirby-Bauer Disk Diffusion Susceptibility Test Send Print

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Created: Tuesday, 08 December 2009
Last update: Monday, 24 June 2013
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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)
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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)  
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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)
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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)
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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)
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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 Hudzicki, University of Kansas Medical Center, Kansas City, KS)
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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)

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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)

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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)

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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)
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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)
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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)

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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)

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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)
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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 Hudzicki, University of Kansas Medical Center, Kansas City, KS)

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