KIRBY-BAUER DISK DIFFUSION TECHNIQUE

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The Kirby-Bauer disk diffusion technique is one of the oldest approaches to the phenotypic antimicrobial susceptibility testing of pathogenic bacteria. This method is widely used for antimicrobial susceptibility test because it is simple and practical to perform, and the technique has been well standardized. Disk diffusion test is a non-automated antimicrobial susceptibility test that still remains one of the most widely used antimicrobial susceptibility testing methods in the routine susceptibility studies of pathogenic microorganisms. It is most suitable for testing the majority of bacterial and fungal pathogens that are commonly encountered in the microbiology laboratory; and it is usually carried out using a template to avoid any form of bias when performing the test (Figure 1).

The template helps the researcher to know the particular spot to place the test antibiotic disk(s) since the disk diffusion technique is not automated for automatic antibiotic disk placement. The Kirby-Bauer disk diffusion test is usually performed using a bacterial inoculum of approximately 1-2 x 108 CFU/ml (obtained or adjusted according to 0.5 McFarland turbidity standards). The test is performed on a Petri dish plate whose size is around 150 mm in diameter, and Mueller-Hinton (MH) agar is recommended for performing this test. The results of the Kirby-Bauer disk diffusion test are “qualitative” in nature – since the susceptibility of the individual organism (usually reported as: susceptible, resistant or intermediate) to the test antibiotic is derived from the disk diffusion test rather than from the minimum inhibitory concentration (MIC). MIC is a more quantitative test than the disk diffusion test that is a qualitative test.  

Briefly, when a paper disk impregnated with a known concentration of a drug is placed on the surface of MH agar plate, moisture from the agar plate is immediately absorbed into the drug laden disk. The drug in the disk begins to diffuse into the surrounding area of the agar. The MH agar plate is incubated at the appropriate temperature (37oC) usually for 18-24 hours or overnight.  Bacterial growth occurs in the presence of an antibiotic when the bacteria reaches a critical mass and can overpower the inhibitory effects of the drug in the paper disk. After incubation, the inhibition zone diameter (IZD) or zones of bacterial growth inhibition around each of the antibiotic (paper) disks are measured to the nearest millimeter (mm) using a meter rule. The IZD measured is related to the antimicrobial susceptibility of the test organism and to the diffusion rate of the antibiotic through the MH agar medium. However, the raw IZD data measured for each of the test organism are usually recorded and interpreted as “susceptible”, “intermediate” and “resistant” category based on the susceptibility of the individual organism to the test antibiotic. 

Figure 1: Template for placement of antibiotic disks on agar plate of 90 mm. Note: When conducting antimicrobial susceptibility testing (AST) in the microbiology laboratory, the distance between antibiotic disks should be 25 mm. This implies that antibiotic disks should be placed at a distance of 25 mm apart. Photo courtesy: https://www.microbiologyclass.com

The interpretation of the disk diffusion test results from IZD to susceptible, intermediate or resistance is done using standard antibiotic breakpoints published by Clinical and Laboratory Standard Institute (CLSI). The point at which critical mass of the bacteria is reached in the disk diffusion technique is demonstrated by a sharply marginated circle of bacterial growth around the paper disk (Figure 2)When the test organism is resistant to the antimicrobial agents used, a smaller zone of inhibition is usually produced (Figure 3). The marginated circle of bacterial growth produced around the antimicrobial disk is often referred to as the inhibition zone of the drug and it is measured to the nearest millimeter using a meter rule. The zone of inhibition is usually compared to standard inhibition zone diameters (antibiotic breakpoints) as per the criteria of CLSI as aforesaid. Generally, the size of the IZD of the drug against the test pathogenic bacteria is used to evaluate the in vivo susceptibility of the organisms to the tested drug. In addition to the inhibition zone diameter (IZD) of a drug to pathogenic bacteria, the phenotypic detection and characterization of antibiotic resistance gene also gives a clue of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the tested drug.

ADVANTAGES OF DISK DIFFUSION TEST

The merits of the disk diffusion test are as follows:

  • Disk diffusion test is very simple to perform
  • The test results can be easily reported and interpreted by both clinicians and laboratory scientists.
  • Disk diffusion test does not require any special equipment to perform.
  • It is flexible.
  • Disk diffusion test has been standardized for testing many bacteria.
  • It is cost-effective and cheap to perform.  

However, the disk diffusion test is not without some demerits. Disk diffusion test lack automation; and thus it cannot be computerized. And it cannot be used to test all fastidious or slow-growing bacteria. The standard antibiotic breakpoints is published and updated by various international committees and bodies including:

  1. Clinical and Laboratory Standard Institute (CLSI).
  2. European Committee on Antimicrobial Susceptibility Testing (EUCAST).
  3. British Society for Antimicrobial Chemotherapy (BSAC). 
Figure 2: Petri dish plate showing zone of inhibition (IZD) by Kirby-Bauer disk diffusion technique. The organism is a clinical isolate of Escherichia coli obtained from the Microbiology Laboratory Department of University of Nigeria Teaching Hospital (UNTH), Ituku-Ozalla, Enugu State, Nigeria. The microbe is susceptible to gentamicin (CN, 10 µg), ofloxacin (OFX, 5 µg), ciprofloxacin (CIP, 10 µg), and sulphamethoxazole-trimethoprim (SXT, 25 µg). Arrow shows the zone of growth inhibition that is to be measured using a meter rule or caliper. Photo courtesy: https://www.microbiologyclass.com
Figure 3: Petri dish showing zone of inhibition caused by antibiotic impregnated disks. The organism is a clinical isolate of Klebsiella pneumoniae from the Microbiology Laboratory of UNTH, Enugu State, Nigeria. The isolate is resistant to ofloxacin (OFX, 5 µg), ciprofloxacin (CIP, 10 µg), and sulphamethoxazole-trimethoprim (SXT, 25 µg). Photo courtesy: https://www.microbiologyclass.com

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

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