5.4.1: Antimicrobial Susceptibility Test — Read

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Antimicrobial susceptibility tests are performed to determine the effectiveness of antimicrobial drugs in inhibiting microbial growth. This is an important test in the clinical lab, especially as microorganisms continue to become resistant to more of the antimicrobial drugs that have traditionally used to treat infections.

Antibiotic resistance occurs when a microorganism is not inhibited or killed by the drug. The resistance occurs as the organism acquires mechanisms to prevent the drug from working. The antimicrobial susceptibility test allows for the determination of which drugs would be potentially effective in treating an infection.

Antimicrobial sensitivity tests are performed by inoculating a plate with a lawn of the bacteria to be assessed and then placing filter discs with known concentrations of drug on the surface of the agar. After incubation, drugs that work to inhibit the growth of the bacteria produce clear zones around the disc known as zones of inhibition. The antimicrobial sensitivity test must be performed using a standardized method so the results obtained are consistent across the healthcare industry. The reason the technique must be standardized is that there are several factors that may affect the size of the zone of inhibition, including the molecular weight of the drug, the type of media used for the test, and the concentration of the bacterial culture. The Kirby-Bauer method is the standardized method employed to ensure reliable results are obtained. This method makes use of a special media known as Mueller-Hinton agar, which is poured to a depth of 4 mm using 100- or 155-mm diameter plates. This is important because it affects how the drug diffuses. If they are poured to thick, then the drug diffusion rate laterally slows resulting in smaller zones of inhibition. To ensure the bacterial concentrations are consistent, they are prepared using 0.5 McFarland standards. Disks using known concentrations of the drug are utilized for the method. The plates are incubated for 16-18 hours. Following incubation, the plate is assessed for zones of inhibition and the zones are measured in millimeters. It is important to note that effectiveness cannot be determined visually. The measurements must be compared to a set of standardized values. In doing so, the measurements will indicate that the microorganism is susceptible (S), intermediate (I), or resistant(R) to the drug. If an organism is determined to be susceptible to the drug, it means that the concentration of the drug usually used is capable of inhibiting the bacteria, and there is good chance that the drug may work to treat the infection in the patient. If the result is intermediate, it means that higher than normal concentrations would be needed to inhibit the bacteria. The effectiveness for using the drug to treat the patient would be uncertain, and that if used for treatment that it would likely only be effective in higher-than-normal doses. A resistant result indicates that the bacteria are resistant in vitro in the lab, and if it is resistant in the lab there is a likelihood that it would be ineffective in treating the infection in the patient.

In this lab you will perform an antimicrobial sensitivity test using the Kirby-Bauer method.

Mueller-Hinton agar plate used in a Kirby-Bauer antibiotic susceptibility test. — “This 1972 image depicts a Mueller-Hinton agar culture plate that had been used in an antibiotic susceptibility test (AST). Known as the Kirby-Bauer method, each of the small, labeled discs, or wafers, contained an antimicrobial agent. This image is an enlarged view of a region of PHIL 10784. The light halos surrounding each disc, also known as reaction zones, represented regions in which the bacteria on the agar’s surface did not thrive, due to their sensitivity to the antibiotic that had been soaked into these respective discs. These tests, which measure the presence or absence of a reaction zone, also take into account the pH of the medium, the concentration of the inoculum, and a number of other factors. Using this method, the sensitivity a bacterial strain has to a given antimicrobial, or combination of antimicrobials, may be determined.”
Source: CDC/ Gilda L. Jones, CDC PHIL, Public domain

Petri dish culture plate showing antibiotic sensitivity test results. — “This Petri dish culture plate contained an unknown growth medium, to which an inoculum of Staphylococci sp. bacteria had been applied. Atop the growth medium, antibiotic-impregnated disks had been laid, which gave rise to this example of an antibiotic sensitivity test. The medium was kept at a pH of 7.2. The size of the inhibition zones surrounding the antibiotic-impregnated paper disks, depended on the level of sensitivity the bacteria, present in the agar, had to the antibiotic, as well as the antibiotic’s ability to diffuse through the culture medium. The overall pH of the medium, in this case 7.2, also played an important role in influencing the results of this antimicrobial sensitivity study.”
Source: CDC, CDC PHIL, Public domain

This page titled 5.4.1: Antimicrobial Susceptibility Test was last modified on Sat, 25 Jul 2026 03:43:16 GMT and is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Sherry Smith.

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