Thedisk diffusion test (also known as theagar diffusion test,Kirby–Bauer test,disc-diffusion antibiotic susceptibility test,disc-diffusion antibiotic sensitivity test andKB test) is aculture-basedmicrobiology assay used indiagnostic anddrug discovery laboratories. In diagnostic labs, the assay is used to determine the susceptibility of bacteria isolated from a patient's infection to clinically approved antibiotics. This allows physicians to prescribe the most appropriate antibiotic treatment.^[1][2][4][5] In drug discovery labs, especiallybioprospecting labs, the assay is used to screen biological material (e.g. plant extracts, bacterial fermentation broths) and drug candidates for antibacterial activity. When bioprospecting, the assay can be performed with paired strains of bacteria to achieve dereplication and provisionally identify antibacterialmechanism of action.^[6][7]

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 antibioticstops the bacteria from growing orkills the bacteria, there will be an area around the disk where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The susceptibility of the bacterial isolate to each antibiotic can then be semi-quantified by comparing the size of these zones of inhibition to databases of information on known antibiotic-susceptible, moderately susceptible and resistant bacteria. In this way, it is possible to identify the most appropriate antibiotic for treating a patient's infection.^[1][2] Although the disk diffusion test cannot be used to differentiate bacteriostatic and bactericidal activity, it is less cumbersome than other susceptibility test methods such asbroth dilution.^[4]

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 anATCC orNCTC strain), and disks containing the test extract are applied to the surface (seebelow).^[6] Zone of inhibition sizes cannot be used as a semi-quantitative measure of antibacterial potency because different extracts contain molecules with different diffusion characteristics (differentmolecular sizes,hydrophilicities etc.). Zone of inhibition sizes can be used for the purpose of dereplication though. This is achieved by testing each extract against paired strains of bacteria (e.g. streptomycin-susceptible and -resistant strains to identify streptomycin-containing extracts). Paired strains (e.g. wild type andtarget overexpressing strains) can also be used to identify antibacterial mechanism of action.^[6][7]

Agar diffusion was first used byMartinus Beijerinck in 1889 to study the effect ofauxins on bacterial growth. However, the method has been developed, refined and standardized by many scientists and scientific organizations over the years including George F. Reddish,Norman Heatley, James G. Vincent,^[8] Alfred W. Bauer, William M.M. Kirby,John C. Sherris,^[4][5] Hans Martin Ericsson, theWorld Health Organization, theClinical and Laboratory Standards Institute, the Swedish Reference Group for Antibiotics, theDeutsches Institut für Normung, theBritish Society for Antimicrobial Chemotherapy and others.^[8]

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 lowerminimum 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]

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 beMueller–Hinton agar at only 4 mm deep, poured into either 100 mm or 150 mm Petri dishes. ThepH level of the agar must be between 7.2 and 7.4. Bacterial inoculum is prepared by diluting a broth culture to match a 0.5McFarland turbidity standard, which is equivalent to approximately 150 millioncells per mL.^[1]

Usingaseptic technique,brothculture of a specific organism is collected with asterileswab. In the case ofGram negative bacteria, excess liquid is removed from the swab by gently pressing or rotating it against the inside of the tube. The swab is then streaked across a Mueller–Hinton agar plate to form a bacterial lawn. To obtain uniform growth, the agar plate is streaked with the swab in one direction, rotated 120° and streaked again, rotated another 120° and streaked again. Using an antibiotic disk dispenser, disks containing specific antibiotics are then applied to the plate. This must be done within 15 minutes of inoculation. Flame-sterilized forceps are used to gently press each disk onto the agar and ensure it is attached. Plates are thenincubated overnight, usually at a temperature of 35 °C. Plates must be incubated within 15 minutes of applying antibiotic disks.^[1]

Certain bacteria require addition of 5% solution of mechanically defibrinated horse blood andβ-NAD (MH-F agar).^[1] The following table shows media requirements of commonly tested microorganisms:

To ensure veracity of test results,quality control methods must be used. In order to monitor the performance of the test, special bacterial strains are used as positive or negative control. When efficacy of β-lactamase is tested, special strains that exhibit β-lactam resistance are used. Additionally, specific media are used to test certain antibiotics. For example, when testingco-trimoxazole susceptibility, media with excessthymine andthymidine are recommended.^[1] The following table lists commonly used quality control strains in the disk diffusion method:

Several variations of the disk diffusion method have been developed including the Oxford penicillin cup andEtest methods used in hospital diagnostic laboratories,^[28][29] and the well diffusion, cylinder diffusion and bioautography methods used in drug discovery and development laboratories.^[6][30]

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.^[31] 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.^[28]

The RAST method serves as a fast means of ascertaining antibiotic susceptibility and was created as a modification of the classic disk diffusion test. It allows to shorten the time of incubation to 16-20 hours. Test scores are read after 4, 6, 8 and 16-20 hours. Compared to the standard method, RAST does not give distinct zones of inhibition within such a short timespan (all bacteria except forS. pneumoniae have a chance of being possible to read after 6 hours higher than 90%). As for quality control strains, they are diluted 1:1 000 000 and defibrinated horse or sheep blood is added. Special RAST breakpoint tables should be used when interpreting the results due to method calibration differences.^[32]

Validated quality control strains include:E. coli ATCC 25922,P. aeruginosa ATCC 27853,S. aureus ATCC 29213,E. faecalis ATCC 29212,S. pneumoniae ATCC 49619.^[33]

RAST allows for rapid determination of possible antibiotic resistance in tested cultures. It allows to check for ESBL and/or carbapenemase producingE. coli andK. pneumoniae, using cefotaxime/ceftazidime (after 4 hours) and meropenem (after 6 hours) respectively. However, these results are not quantitative and should be used only for screening in routine medical tests.^[34][35] In clinical trials, RAST method led to significant improvements in predicting efficacy of antibiotic therapy.^[36]

Disks containing antibiotics are placed on an uninoculated Mueller-Hinton agar plate and incubated for 2 hours. Then, they are removed and the bacterial suspension previously prepared using broth microdilution is applied and another disk with a different antibiotic is placed precisely in the same place as the previous one. After incubation for 16-20 hours results are correlated with the first antibiotic's MIC values. An example of the pre-diffusion method is testingin vitro efficacy of ceftazidime/avibactam (primary disks) in terms of aztreonam (secondary disks).^[37]

Disk diffusion method can be used to test susceptibility to antifungals.^[38][39]

Comparing to classical disk-based methods, bioautography utilisesthin-layer chromatography to separate constituents of the tested mixture. Then, the TLC plate can be either placed on the inoculated agar and be allowed to diffuse into it (contact bioautography) or be covered with microbe-containing broth (direct bioautography). Then, the sample is incubated and zones of inhibitions are measured.^[41][42][40] Alternatively, the TLC plate can be covered with molten agar in theagar overlay bioautography.^[43]

To visualise zones of inhibitions in direct bioautography, reagents that detectdehydrogenase activity are used (e.g.,tetrazolium salts, which are converted by microbial dehydrogenases into chromogenicformazans).^[41]

• 
  Agar diffusion was first used in 1889 byMartinus Beijerinck.^[8]
• 
  A close-up look at the results of an agar diffusion test
• 
  An antibiogram ofSerratia marcescens. Each disk is labelled with the antibiotic it contains (e.g. AMC30, 30 μgamoxicillin/clavulanic acid)
• 
  Automated culture plate reader to measure inhibition zone diameters

• Antibiotic sensitivity testing
• Double-disk diffusion test
• Etest

• Microbiology techniques
• Antimicrobial resistance

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