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Literature Review: factors in selecting drugs in Zone of Inhibition (part 1)

Updated on June 9, 2011


A variety of laboratory methods can de used to measure in vitro susceptibility of bacteria to antimicrobial agents in vitro. Among them the Kirby Bauer disk diffusion test is one of the standardized methodologies recommended by the Clinical and laboratory standards institute (CLSI) and US Food and Drug Administration (FDA). This method relies on the inhibition of bacteria growth around varies antimicrobial impregnated filter paper disks measured under standard antibiotic resistance conditions. When performed and evaluate carefully, this method has been extremely useful as a guide in choosing an antimicrobial agent best suitable for a bacterial disease and to evaluate the antibacterial efficiency. The purpose of this paper is to characterize the Kirby Bauer disk diffusion test and the potential application of the test in different sectors. This paper also discusses existing and previous scientific research using a standard procedure or modified form of the procedure of .


The Kirby-Bauer test, known also as the disk-diffusion method, is the most widely used for routine antibiotic susceptibility test based solely on the presence or absence of a zone of inhibition without regard to the in determine what treatment of antibiotics should be used when treating an infection. In this test, an agar plate is uniformly inoculated with the test organism and a paper disk impregnated with a fixed concentration of an antibiotic is placed on the agar surface. Growth of the organism and diffusion of the antibiotic commence simultaneously resulting in a circular zone of inhibition in which the amount of antibiotic exceeds inhibitory concentrations. The diameter of the inhibition zone is a function of the amount of drugs in the disk and susceptibility of the microorganism.

What is zoned of inhibition?

It is the area on an agar plate where growth of a control organism is prevented by an antimicrobial agent (antibiotic,etc)usually placed on the agar surface in disk diffusion test. If the test organism is susceptible to the antibiotic, they will not grow near the disk. If the organism is resistant to the antibiotic, they will grow right up to the disk. The size of the zone is proportional to how sensitive the organism to the antibiotic.

History behind other zone of inhibition:

The isolation of penicillin from Penicillium natatumbyAlexander Fleming in1929 Isa's milestone in the history of medicine. As more antimicrobial compounds were discovered. It was predicted that infectious diseases would be eliminated through the use of these antimicrobials. Unfortunately, the development of bacterial resistance to these antimicrobials quickly diminished this optimism and resulted in the need for physicians to request the microbiology lab to test a patient’s pathogen against various concentrations of given antimicrobialtodeterminesusceptibility orresistance tothat drug. The original method of determining susceptibility of Mia's microorganisms was based on broth dilution methods, which although still the reliable standard today. BuHowever,he method is time consuming to perform. This prompted the development of disk diffusion procedure for the determination of bacteria otantritic. By the early 1950s, most clinical microbiology laboratories in the United States had adopted the disk diffusion method for determining susceptibility of bacteria otantritic. Each lab modified the procedure to suit its own needs. Besides many researchers published variations for the procedure resulting in multiple protocols that resulted from in widespread confusion. In1956, W.M.M.Kirby and his colleagues at the University OfWashington School OfMedicine and the King County Hospital proposed a single disk method for antimicrobial susceptibility testing. However, he lacked of standardization for the determination of bacterial susceptibility continued to be a problem throughout the early 1960s. Kirby and his colleague, A.W.Bauer, extensively reviewed the susceptibility testing literature on antibiotic resistance. They consolidated and updated all the previous descriptions of the disk diffusion method and published their findings. This publication led the World Health Organization to form a committee in1961 to lay the groundwork for the development of standardized procedure for single antimicrobial disk susceptibility testing. The result was a standardized procedure for the disk diffusion susceptibility test, henceforth called the Kirby-Bauer disk diffusion test.

Figure 1: Bacterial growth in agar plate and measurement of Zone diameter

Materials and methods

Materials required:

Petridish, forceps, paper disk, distilled water, micropipette and tips.

The reagents required for the method are given below:

Nutrient agar media, bacterial culture, antibiotics, ethanol.


1. Preparation of nutrient agar plates

Nutrient agar plates with appropriate turbidity are prepared by pouring plate method. Mueller-Hinton agar is considered the best medium to use for routine susceptibility testing of non-fastidious bacteria. Fastidious organisms require Mueller-Hinton agar supplemented with additional nutrients.

2. Preparation of inoculum

A sterile inoculation loop or needle swap in the bacterial suspension and the loop is streaked in at least three directions over the surface of the nutrient agar to obtain uniform growth. A final sweep is made around the rim of the agar. The plates are allowed to dry for approximately five minutes.

3. Placement of the antibiotic disks

Disks containing the test antibiotic is placed on the surface of the agar, using either flame-sterile forceps to dispense each antibiotic disk one at a time, or a multidose dispenser to dispense multiple disks at one time. The distribution of the disks should apart from each other and not close to the edges of the plate.

4. Incubation of the plates

The plates are incubated within 15 minutes after applying the disks. The plates should be incubated soon after placing the disk. The temperature range of 35° ± 2°C is normally required for incubation and incubation time is not less than 28 hours but the 28 hours are considered as standard for this test.

5. Measuring zone diameter

After the plates have been overnight incubated, there should be a noticeable “clearing zone” around each of the antibiotic discs. The diameter of the each disk is measured and recorded in millimeters (mm). For measure, the plates are carefully examined for well-developed colonies within the zone of inhibition.

6. Interpretation of the results:

After measurement, each measurement can be compared to a zone-size interpretive chart. Using the chart, the organism can be characterized as being resistant, intermediate or susceptible to the specific antibiotic resistance. Intermediate susceptibility means that some inhibition from the antibiotic occurred but not sufficiently enough to inhibit the growth of the organism in the body. Antibiotic placed medium surface should be considered.


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      solomon samuel 5 years ago

      i would like to say thanks to the author for making this susceptibility test a simple and self explanatory. Thanks a lot

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      subin 6 years ago

      thanks for this review

    • ankandhk profile image

      ankandhk 6 years ago from A blessing from Almighty God!

      Thanks for your comment.

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      Giselle Maine 6 years ago

      A clear and helpful explanation of this science technique. Thanks for writing on this unique topic.