How to test antibiotic activity? A Guide to Key Laboratory Methods

October 12, 2025 •

3 min read

According to the World Health Organization, antibiotic resistance is one of the top ten global public health threats. As a result, understanding how to test antibiotic activity is critical for both clinical treatment and the discovery of new antimicrobial agents. These standardized laboratory methods are used to determine a pathogen's susceptibility to antibiotics, guiding effective therapy and tracking emerging resistance patterns.

Quick Summary

This guide explains the primary methods for assessing antibiotic activity in the laboratory, from standard culture-based techniques like disk diffusion and broth dilution to more advanced and automated systems. It covers key metrics such as Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), explaining how these tests inform clinical practice and antimicrobial research.

Key Points

Kirby-Bauer Method: This disk diffusion test provides a qualitative assessment of an antibiotic's effectiveness by measuring the zone of inhibition around a disc on an agar plate.
Broth Dilution Method: Considered the gold standard, this method offers a quantitative measure of antibiotic efficacy, determining the Minimum Inhibitory Concentration (MIC) in a liquid medium.
Minimum Inhibitory Concentration (MIC): The MIC is the lowest concentration of an antibiotic that prevents the visible growth of a specific microorganism.
E-test (Gradient Diffusion): This technique uses a pre-calibrated plastic strip with a concentration gradient to determine the MIC with greater precision than disk diffusion.
Clinical Significance: Accurate susceptibility testing is essential for selecting the correct treatment for patients, optimizing dosages, and monitoring the rise of antibiotic resistance.
Advanced Techniques: Beyond traditional culture methods, techniques like automated systems, molecular diagnostics, and time-kill assays offer faster results and greater insight into antimicrobial action.

The Importance of Testing Antibiotic Activity

Antimicrobial susceptibility testing (AST) is an essential function in modern medicine and pharmacology. Before prescribing a medication for a bacterial infection, clinicians must know which antibiotics will be effective against the specific pathogen. The rise of multi-drug resistant (MDR) bacteria has made this testing more critical than ever, as standard treatments may fail, leading to more severe infections and higher mortality rates. In research, testing antibiotic activity is vital for screening new compounds and monitoring trends in resistance. These tests provide information that guides the selection of the correct drug, optimizes dosages, and helps track resistance for public health surveillance.

Standard Phenotypic Methods

The most widely used methods for testing antibiotic activity are based on observing a microbe's growth (phenotypic testing) when exposed to a specific drug.

Disk Diffusion (Kirby-Bauer) Assay

One of the simplest and most cost-effective methods, the Kirby-Bauer test provides a qualitative assessment of antimicrobial susceptibility. It involves:

Spreading a standardized bacterial suspension on a Mueller-Hinton agar plate.
Placing paper discs containing antibiotics on the agar.
Incubating the plate, allowing antibiotics to diffuse.
Measuring the diameter of the clear 'zone of inhibition' where bacterial growth is prevented.

Comparing zone diameters to standard charts determines if the bacteria are susceptible (S), intermediate (I), or resistant (R). While simple and inexpensive, it doesn't provide a precise MIC and is affected by diffusion rates.

Broth Dilution Method

Considered a gold standard, this method quantifies the Minimum Inhibitory Concentration (MIC), the lowest antibiotic concentration inhibiting visible growth. The procedure typically involves preparing serial dilutions of the antibiotic in broth, inoculating with bacteria, and incubating. The MIC is the lowest concentration showing no visible turbidity. This method can also determine the Minimum Bactericidal Concentration (MBC), the lowest concentration that kills most bacteria, by sub-culturing from wells with no growth onto antibiotic-free media. Broth dilution is quantitative and reproducible but more labor-intensive than disk diffusion.

Antimicrobial Gradient Method (E-test)

Combining diffusion and dilution principles, the E-test uses a strip with a pre-defined antibiotic concentration gradient placed on a bacterial lawn on an agar plate. An elliptical inhibition zone forms after incubation, and the MIC is read where the zone intersects the strip's scale. This method provides a precise MIC, is adaptable, and easy to perform, though more expensive per test strip than disc diffusion.

Advanced and Specialized Techniques

Beyond standard methods, advanced techniques offer faster or more specific analysis:

Automated Systems: Platforms like VITEK or Microscan use panels with dried antibiotics to automatically track bacterial growth changes and provide MICs or susceptibility categories within hours.
Molecular and Genetic Methods: Techniques such as PCR and genome sequencing identify resistance genes, offering rapid results but limited to known mechanisms.
Time-Kill Kinetics: This research method tracks viable bacteria counts over time after antibiotic exposure to create a 'kill curve,' showing the rate of killing or inhibition.
ATP Bioluminescence Assay: A rapid method measuring ATP from live cells; decreased luminescence indicates reduced viability and antimicrobial activity.

Comparison of Antibiotic Activity Testing Methods


Feature	Disk Diffusion (Kirby-Bauer)	Broth Dilution (MIC/MBC)	Antimicrobial Gradient (E-test)	Automated Systems
Measurement	Qualitative (S/I/R)	Quantitative (MIC/MBC)	Quantitative (MIC)	Quantitative (MIC/S/I/R)
Principle	Agar diffusion from disc	Serial dilution in broth	Concentration gradient on strip	Optic measurement in panels
Information	Zone of inhibition diameter	Lowest concentration to inhibit growth	Intersection of ellipse with strip	Automated reading of growth
Speed	16–18+ hours	16–24+ hours	16–18+ hours	Hours
Cost	Low	Low to Moderate	High (per strip)	High (capital cost)
Throughput	High (many discs/plate)	High (microplate format)	Moderate	Very high
Complexity	Low	High (manual)	Moderate	Low (after setup)

The Clinical and Public Health Impact

Accurate antibiotic testing is vital for patient care, ensuring effective treatment and preventing the use of ineffective drugs that contribute to resistance. On a larger scale, testing provides surveillance data crucial for tracking resistant organisms and informing public health responses. Organizations like the European Committee on Antimicrobial Susceptibility Testing (EUCAST) provide guidelines to standardize these procedures globally. Advancements in rapid diagnostics promise faster results for critical treatment decisions.

Conclusion

Testing antibiotic activity is fundamental to managing infectious diseases and developing new drugs. Methods range from the simple Kirby-Bauer test to precise broth dilution and automated systems. As antimicrobial resistance grows, refining current techniques and developing faster diagnostics are essential in combating infectious diseases.

For more detailed guidelines, refer to the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

Frequently Asked Questions

The Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism in a laboratory setting.

MIC is the lowest concentration of an antibiotic that inhibits visible growth, meaning it is bacteriostatic. The Minimum Bactericidal Concentration (MBC) is the lowest concentration that kills 99.9% of the initial bacterial population.

In the Kirby-Bauer test, a uniform lawn of bacteria is grown on a plate. Discs containing antibiotics are placed on the surface. After incubation, the diameter of any clear zones where growth is inhibited is measured and compared to a standard chart.

Testing antibiotic activity is crucial for selecting the most effective treatment for a patient's infection, preventing the use of ineffective drugs, and monitoring trends in antibiotic resistance at a public health level.

The E-test, or antimicrobial gradient diffusion test, is used to determine the exact Minimum Inhibitory Concentration (MIC) of an antibiotic against a microorganism by creating a concentration gradient on an agar plate.

Results can be affected by factors such as the type of growth medium, inoculum density, incubation time and temperature, and the specific test technique used.

The zone of inhibition is the area of no bacterial growth around an antibiotic disc in a disk diffusion test. Its size is used to determine if a bacterium is susceptible, intermediate, or resistant to the antibiotic.