Methods to Test Susceptibility of Microorganisms to Antibiotics

Species of microorganisms exhibit divergence in degrees of susceptibility to different antibiotics. In addition to this, an organism’s susceptibility to a given antibiotic may change. This is especially true during treatment. For the most satisfactory results in treatment, a clinician has to know the identity of the microorganism and the specific antibiotic he will use.

The services of a microbiologist are necessary to assist the clinician along this line. The microbiologist will make an accurate microbiological diagnosis and determine the susceptibility of the organism to various antibiotics. During the course of therapy, the microbiologist may, from time to time, be required to make appraisals on any change in the susceptibility of the pathogen to the antibiotic. He may likewise perform an analysis of the antibiotic concentration in the body fluids.

There are several methods by which the susceptibility of microorganisms to antibiotics (as well as to other chemotherapeutic agents) can be determined. Two of these microbial susceptibility tests are the tube dilution technique and the paper disk plate method.

Using the tube dilution technique, the microbiologist is able to determine the smallest amount of antibiotic required to inhibit the growth of the microorganism outside the living body and in an artificial environment. The amount is called minimal inhibitory concentration (MIC). In the tube dilution microbial susceptibility test, increasing amounts of the antibiotics being examined are placed in a series of culture tubes containing a suitable broth medium into which the microorganism being tested was introduced. After incubation, the concentration of antibiotics necessary to inhibit the growth of the organism used is determined by observing the absence of growth.

The most commonly used method for determining susceptibility of microorganisms to antibiotics is the paper disk plate technique. Small paper disks permeated thoroughly with known amounts of commercially available antibiotic are placed upon the surface of an inoculated plate. After inoculation, the plates are inspected for any clear areas (or zones of inhibition) surrounding the disks. A clear area around the disk is an indication that the organism was inhibited by the antibiotic, which diffused into the culture medium from the disk.

The Kirby-Bauer antibiotic testing, which was developed in 1966, is a slight modification of the paper disk plate microbial susceptibility test. This is a highly-standardized single-disk technique, in which the amount of antibiotic contained in the disk is specified as well as at least three other details: the test medium, size of the material used for inoculation, and conditions of incubation.


1. “Micromethod (Spot-Plate) Determination of In Vitro Antibiotic Susceptibility” by R. A. Beargie, E. C. Bracken, and H. D. Riley, Jr., Department of Pediatrics, Children’s Memorial Hospital, University of Oklahoma Medical Center, Oklahoma City, Oklahoma –

2. Ligeza, J (J), “Susceptibilityto nitrofurantoin, biseptol and nalidixic acid of E. Coli strains isolated from urine in the years 1983-1990” –

3. “Susceptibility Testing,” on The Merck Manuals Online Medical Library –