Antimicrobial testing encompasses a range of laboratory procedures designed to evaluate how effective antimicrobial agents are against specific microorganisms, such as a bacterium or fungus. The primary goal is to determine if a microbe is affected by an antimicrobial or if it possesses resistance. This information is used to guide physicians in selecting appropriate drug therapies, assist the pharmaceutical industry in drug discovery, and help public health organizations monitor the spread of antimicrobial resistance.
Diffusion-Based Assays
Diffusion-based assays work by allowing an antimicrobial agent to move from a concentrated source through a solid growth medium, like agar, seeded with a test microorganism. As the agent disperses, its concentration decreases with distance, creating a gradient. If the microorganism is susceptible, its growth is prevented in areas where the antimicrobial concentration is high enough, resulting in a clear area.
The most recognized example is the Kirby-Bauer disk diffusion test. In this procedure, small paper disks saturated with a known antibiotic concentration are placed on an agar plate uniformly inoculated with the test bacterium. After incubation, a “zone of inhibition” appears as a clear circle where the antibiotic concentration was high enough to stop bacterial growth. The diameter of this zone is measured and compared to standardized charts to classify the organism as susceptible, intermediate, or resistant.
Variations of this technique offer different advantages. The agar well diffusion method involves punching a hole in the agar and filling it with a liquid antimicrobial agent instead of using a disk. The gradient diffusion method uses a plastic strip (such as an E-test) containing a predefined, continuous antibiotic gradient. When placed on an inoculated plate, this strip creates an elliptical zone of inhibition, and the point where the zone edge intersects the strip indicates a precise quantitative value.
Dilution Methods for Susceptibility Testing
Dilution methods provide a more quantitative assessment of an antimicrobial’s effectiveness by exposing microorganisms to a series of decreasing concentrations of the agent. Unlike diffusion assays that rely on observing a zone of no growth, dilution tests pinpoint the specific concentration required to inhibit the organism. This is achieved by preparing a range of antimicrobial concentrations in a liquid broth or solid agar medium, which is then inoculated with the test microbe.
The most common application is the broth dilution method, performed in test tubes (macrodilution) or, more frequently, in small microtiter plates (microdilution). The microdilution format is widely used in clinical laboratories because it allows for the simultaneous testing of multiple antibiotics against a single microbe in a compact plate. Another approach is agar dilution, where the antimicrobial is mixed directly into the molten agar before it solidifies, creating a series of plates with different drug concentrations.
These methods are used to determine the Minimum Inhibitory Concentration (MIC). The MIC is defined as the lowest concentration of an antimicrobial drug that prevents the visible growth of a microorganism after incubation. In broth dilution tests, this is identified by observing the first well in the series that remains clear. The MIC value provides a precise measure of an antimicrobial’s potency against a specific pathogen.
Determining Bactericidal Activity
While the Minimum Inhibitory Concentration (MIC) reveals the concentration needed to stop microbial growth, it does not distinguish between agents that inhibit growth (bacteriostatic) and those that kill organisms (bactericidal). This distinction is important in medical contexts, such as treating infections in patients with weakened immune systems. For these situations, it is necessary to ensure the pathogen is eradicated rather than just suppressed.
To assess killing activity, the Minimum Bactericidal Concentration (MBC) is determined. The MBC is defined as the lowest concentration of an antimicrobial agent that kills a specified percentage, typically 99.9%, of the initial bacterial population. Determining the MBC is a direct extension of the MIC test, involving taking a sample from the clear wells of the completed MIC assay and transferring it to a fresh agar plate containing no antimicrobial agent.
The new plates are incubated to see if any viable bacteria from the MIC test grow. If bacteria appear, the corresponding concentration was only bacteriostatic. The lowest concentration from the MIC series that results in no growth on these subculture plates is identified as the MBC, providing a measure of the drug’s lethal effect.
Modern and Rapid Testing Approaches
The need for faster results has driven the development of advanced methods that move beyond traditional culturing. Automated systems, like VITEK, BD Phoenix, and MicroScan, are common in clinical laboratories. These instruments automate the incubation, reading, and interpretation of susceptibility tests, often using miniaturized broth microdilution. They monitor microbial growth through turbidimetric (cloudiness) or colorimetric (color change) detection, reducing turnaround times from days to hours and improving standardization.
Molecular methods offer a rapid alternative by detecting the genetic basis of resistance directly from a sample, sometimes bypassing the need for culturing. Techniques like the Polymerase Chain Reaction (PCR) can identify specific resistance genes, such as those that inactivate penicillin-like antibiotics. DNA microarrays and whole-genome sequencing provide a more comprehensive view, capable of detecting numerous resistance markers and mutations simultaneously, offering deep insights into an organism’s resistance profile.
Other technologies are also accelerating the process. Mass spectrometry, specifically MALDI-TOF MS, can identify a microorganism within minutes from a single colony. While primarily an identification tool, its speed helps clinicians make quicker, more informed decisions about initial antimicrobial therapy. Coupling these rapid identification methods with advanced susceptibility testing is transforming the management of infectious diseases.