KIRBY-BAUER DISC DIFFUSION
INTRODUCTION
Sensitivity testing by the disc diffusion technique is the most commonly used method of determining susceptibility to antimicrobial agents. Accurate and precise results can be obtained provided that the procedure is carefully standardised and controlled.
The antimicrobial agents are applied to the agar plates in the form of filter paper discs impregnated with a suitable concentration of antimicrobial. Once in contact with the agar, the discs absorb water, which dissolves the antimicrobial in the disc. The agent is then free to diffuse away from the disc into the agar, forming a concentration gradient as it does so. If the plate is incubated at a suitable temperature, the bacteria on the surface of the plate begin to proliferate while diffusion is occurring. When the bacteria reach logarithmic phase, bacterial multiplication proceeds more rapidly than antimicrobial diffusion and in areas where the concentration of antimicrobial is below the minimal inhibitory concentration (MIC), the bacteria continue to grow and a visible lawn of growth appears. In areas where the concentration is greater than the MIC, no growth will occur and a circular "zone of inhibition" will develop around the disc; generally the more susceptible the test organism, the larger the zone. The size however, varies depending on antimicrobial.
The disc diffusion method has been standardised for the testing of rapidly growing, non-fastidious organisms (i.e. those that grow overnight on media containing no added blood or other complex nutritional factors). As development of the zone of inhibition depends to some extent on the rate of growth of the organism, the method is not suitable for slow growing organisms, which develop falsely large zones.
The method most commonly used is based on that of Kirby and Bauer and complies with the criteria set by the Clinical and Laboratory Standards Institute (CLSI at http://www.clsi.org) and Eucast (http://www.eucast.org/) for routine sensitivity testing:
Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Turck. 1966
“Antibiotic Susceptibility Testing by Standardised Single Disc Method”
Am. J. Clin Path. 45:493-496
PROCEDURE
- Pick up 4-5 well isolated colonies of an organism to be tested and inoculate into 5ml tryptone soy broth (TSB).
- Incubate 2-5 hours until visible growth appears.
- Adjust the concentration of the culture to the equivalent of 1/2 a McFarland turbidity standard number 1.
- Inoculate one Mueller-Hinton plate per organism :
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- Dip a sterile cotton swab into the adjusted culture and remove excess fluid by rotating the swab against the side of the container, above the level of the culture.
- Streak the swab over the entire surface of the plate in 3 directions and then make a final sweep around the rim of the plate.
5. Allow to dry for 3-5 minutes, but place the antibiotic discs on the plate within 15 minutes. Position the discs evenly around the plate, at least 15 mm from the edge. No more than 6 discs can be tested on a standard petri plate.
6.Press the discs gently into the surface, invert the plate and incubate immediately at 37oC for 18 hours.
7. Measure the zones of complete inhibition to the nearest mm, and by reference to the standard table (CLSI), determine if each organism is sensitive, resistant or intermediate to the agents tested. These tables are available in the laboratory only. As they are commercial products, we cannot supply them on this website.
8. Alternatively, consult Eucast information at http://www.eucast.org/ .
Disc diffusion video
DIAGNOSTIC SENSITIVITY TESTING
For some organisms, sensitivity to certain antibiotics is a diagnostic criterion. The tests are normally carried out in the same way as a Kirby-Bauer disc diffusion test i.e. the organism is spread on a plate and a disc of antibiotic applied. For most of the tests, any zone of inhibition is considered sensitive.
Often the disc will be placed on primary isolation plates. Organism concentration and choice of media is not considered as crucial as in a sensitivity test used to direct clinical treatment.
Bacitracin
S. pyogenes (Lancefield group A) is sensitive, all other beta-haemolytic Streptococci are resistant.
Optochin
S. pneumoniae is sensitive, all other alpha-haemolytic Streptococci are resistant.
Novobiocin
S. epidermidis is sensitive, S. saprophyticus is resistant (both are coagulase negative Staphylococci common in UTI).