Modified Kirby-Bauer Disc Diffusion Method

Modified Kirby-Bauer disc diffusion test method is a reference method that could be used as a routine technique to test the susceptibility of a bacterial isolate in a clinical laboratory. The disc diffusion method was originally described in 1966, is well standardized, and has been widely evaluated.

Procedure for Modified Kirby Bauer method

1. Prepare the inoculum from the primary culture plate by touching with a loop the tops of each of 3 – 5 colonies of similar appearance*, of the organism to be tested and transfer this growth to a tube of saline. If the inoculum has to be made from a pure culture,  suspend a loopful of the confluent growth similarly.
   *Note: Multiple similar looking colonies should be picked to minimize the possibility of testing a non representative colony such as picking a susceptible colony only and missing the resistant mutants dispersed in other colonies.
2. Compare the tube with the 0.5 McFarland turbidity standard (approx cell density 1.5 x10^8 CFU/ml) and adjust the density of the test suspension to that of the standard by adding more bacteria or more sterile saline.
   Remember: Proper adjustment of the turbidity of the inoculum is essential to ensure that the resulting lawn of growth is confluent or almost confluent.
3. Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid.
4. Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60 ° after each application. Finally, pass the swab around the edge of the agar surface. The swab should follow as it is drawn across the plate (as shown in the figure). #Discard the swab into an appropriate container.
5. Leave the inoculum to dry for a few minutes (at least 3 to 5 minutes, but no more than 15 minutes) at room temperature with the lid closed.
6. Place the appropriate antimicrobial-impregnated disks on the surface of the agar (antimicrobial disks can be purchased from any reputable suppliers)
7. Antimicrobial discs can be placed on the inoculated plates using a pair of sterile forceps. It is convenient to use a template to place the discs uniformly or a sterile needle-tip may also be used to place the antibiotic discs on the plate. Alternatively, an antibiotic disc dispenser (as shown in image-2) can be used to apply the discs to the inoculated plate.
8. Disks should not be placed closer than 24 mm (center to center) on the Muller Hinton agar plate.  Ordinarily, no more than 12 disks should be placed on a 150-mm plate or more than 5 disks on a 100-mm plate. #avoid placing disks close to the edge of the plate as the zones will not be fully round and can be difficult to measure.
9. Each disc should be gently pressed down to ensure complete contact with the agar surface and do not fall when the plate is inverted during incubation. #Do not push the disc into the agar.
10. The plates should be placed in an incubator at 35 °C within 30 minutes of preparation. Temperatures above 35 °C invalidate results for oxacillin/methicillin. #Do not incubate in an atmosphere of carbon dioxide, this will decrease the pH of the agar and result in errors due to incorrect pH of the media.
11. After overnight incubation, the diameter of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted according to the antimicrobial susceptibility interpretation chart.

Measurement of the zone of inhibition

The measurements can be made

• with a ruler on the under-surface of the plate without opening the lid.
• If the medium is opaque, the zone can be measured by means of a pair of calipers.
• A template may be used to assess the final result of the susceptibility tests.

The endpoint of inhibition is judged by the naked eye at the edge where the growth starts, but there are three exceptions:

1.  With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone; such growth should be ignored.
2. When β-lactamase-producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of the size of the zone of inhibition, they should be reported as resistant.
3. Certain Proteus species may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored.

Results

Results can be read after 18 hours of incubation. Following incubation, measure the zone sizes to the nearest millimeter (mm) using a ruler or caliper; include the diameter of the disk in the measurement.

Interpretation and Reporting of antimicrobial susceptibility results

1. Using the published CLSI guidelines, determine the susceptibility or resistance of the organism to each drug tested. Note that there are different charts for different organisms.
2. For each drug, indicate on the recording sheet whether the zone size is susceptible (S), intermediate (I), or resistant (R) based on the interpretation chart. Zone sizes are not reported to physicians.

Limitations 

If performed precisely according to standard protocol, disk diffusion method yields data that can reliably predict the in vivo effectiveness of the drug in question but it has a few limitations too:

1. Does not provide accurate information about the minimum inhibitory concentration (MIC)
2. Does not provide reliable results with some antibiotic/organism combinations, such as for penicillin G in Neisseria meningitidis and S. pneumoniae.

References and further reading 

1. “Microbiology A Laboratory Manual” by Natalie Sherman James G. Cappuccino.