Disk diffusion test
The disk diffusion test (also known as the agar diffusion test, Kirby–Bauer test, disc-diffusion antibiotic susceptibility test, disc-diffusion antibiotic sensitivity test and KB test) is a
In diagnostic laboratories, the test is performed by inoculating the surface of an agar plate with bacteria isolated from a patient's infection. Antibiotic-containing paper disks are then applied to the agar and the plate is incubated. If an antibiotic
In drug discovery labs, the disk diffusion test is performed slightly differently than in diagnostic labs. In this setting, it is not the bacterial strain that must be characterized, but a test extract (e.g. a plant or microbial extract). The agar plate is therefore inoculated with a bacterial strain of known phenotype (often an
History
Agar diffusion was first used by
Principle
A pure bacterial culture is suspended in saline, its turbidity is standardized, and it is swabbed uniformly across an agar plate. An antibiotic- or extract-impregnated filter paper disk is then placed on the surface of the agar. The disk constituent(s) diffuse from the filter paper into the agar. The concentration of these constituents will be highest next to the disk and will decrease as the distance from the disk increases. If the antibiotic or extract is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. In general, larger zones of inhibition correlate with lower minimum inhibitory concentrations (MICs) of antibiotic or extract for that bacterial strain.[1] An exception to this is when molecules of the antibiotic or extract are large or hydrophobic because these diffuse through the agar slowly.[6]
Standard method
Agar plate and inoculum preparation
All aspects of the Kirby–Bauer procedure are standardized to ensure consistent and accurate results. Because of this, a laboratory must adhere to these standards. The media used in Kirby–Bauer testing must be Mueller–Hinton agar at only 4 mm deep, poured into either 100 mm or 150 mm Petri dishes. The pH level of the agar must be between 7.2 and 7.4. Bacterial inoculum is prepared by diluting a broth culture to match a 0.5 McFarland turbidity standard, which is equivalent to approximately 150 million cells per mL.[1]
Inoculation and incubation
Using
Alternate methods
Several variations of the disk diffusion method have been developed including the Oxford penicillin cup and Etest methods used in hospital diagnostic laboratories,[9][10] and the well diffusion, cylinder diffusion and bioautography methods used in drug discovery and development laboratories.[6][11]
Oxford penicillin cup method
Disks containing increasing antibiotic concentrations are placed on a seeded bacterial lawn on the agar surface and plates are incubated. Zone sizes are measured from the edge of the disk to the end of the clear zone. Interpretation is more complicated in mixed susceptibility populations. These are plotted as linear dimensions or squares of distances as a function of the natural logarithm of antibiotic concentration in the disks. The MIC is determined from the zero intercept of a linear regression fit through the data.[12] The intercept itself is the logarithm of the MIC. The slope of the regression line is related to the diffusion coefficient of that particular antibiotic in the agar.[9]
Other images
-
Agar diffusion was first used in 1889 by Martinus Beijerinck.[8]
-
A close-up look at the results of an agar diffusion test.
-
An antibiogram of Serratia marcescens. Each disk is labelled with the antibiotic it contains (e.g. AMC30, 30µg amoxicillin/clavulanic acid)
See also
References
- ^ a b c d e f EUCAST (January 2021). "Antimicrobial susceptibility testing: EUCAST disk diffusion method" (PDF). www.eucast.org. EUCAST. Retrieved March 16, 2021.
- ^ PMID 1214010.
- ^ Sahu, BK (2013). Antimicrobial properties of aerial part of Sesbania grandiflora (Linn.) (Semester project). The Pharmaceutical College Barpali, India.
- ^ PMID 13669774.
- ^ PMID 5325707.
- ^ S2CID 254932190.
- ^ S2CID 986144.
- ^ PMID 11420332.
- ^ PMID 18339637.
- S2CID 204029543.
- S2CID 226257747.
- ^ "Analysis of bacterial sensitivity to antibiotics by the agar diffusion method". agardiffusion.com. Retrieved March 16, 2021.