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Zone of Inhibition Test
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Determine the lowest concentration of an antimicrobial agent that visibly inhibits the growth of a microorganism
Determine the lowest concentration of an antimicrobial agent that kills 99.9% of the initial bacterial population.
Quick understanding of the test
Zone of Inhibition Test
Application
- Bacterial culture is spread on Mueller-Hinton agar.
- The plate is incubated for 18-24 hours with the treated product.
- The zone of inhibition around the product is measured; larger zones indicate stronger antibacterial activity.
- Provides quick results, helping researchers efficiently assess antimicrobial potential.
- Offers clear, measurable results through the visible inhibition zones around the tested sample.
Turnaround Time
The test typically takes 4 weeks to complete.
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Abstract
The Zone of Inhibition Test is also known as the Kirby-Bauer Test, Disk Diffusion Test, Agar Diffusion Test, or Antimicrobial Susceptibility Test. It is a simple, inexpensive and quick method to evaluate the antimicrobial effectiveness of a substance or material against specific microorganisms.
Originally developed in the 1950s to measure antibiotic resistance in clinical isolates, this method was standardized by the World Health Organization in 1961 and has since become a valuable screening method in the medical device, pharmaceutical, and material science industries.
The ZOI test is particularly useful for evaluating leachable antimicrobial agents embedded in plastics, textiles, coatings, gels, liquids, and other solid or semi-solid products. Its rapid turnaround makes it an excellent preliminary tool for screening and comparing antimicrobial performance across different formulations or product designs.
How Is a Zone of Inhibition Test Performed?
- A pure culture of the target bacterial or fungal strain is prepared.
- Using a sterile swab, the test suspension is evenly spread over Mueller-Hinton agar plates.
- Using sterile forceps, the treated product sample is placed in the centre of the agar plate.
- The agar plate is then incubated for 18-24 hours at 36ยฐC along with other optimal conditions for bacterial growth.
- After the incubation period, the plate is examined for the presence of a clear area (zone of inhibition) around the antibacterial product sample. If present, the zone of inhibition is measured.
- The size of ZOI correlates with the potency of the antimicrobial agent in the treated product. The higher the antibacterial activity, the larger the zone is and vice versa.
Applications of Zone of Inhibition test
- Clinical microbiology: ZOI tests help determine how effective antibiotics are for treating bacterial infections and track the emergence of antibiotic resistance.
- Antimicrobial Susceptibility Testing (AST): ZOI assays show whether bacteria are sensitive to a specific antimicrobial. If the bacteria are susceptible, clear zones appear where growth is prevented. Sometimes, partial zones form if the effect is incomplete.
- Minimum Inhibitory Concentration (MIC): ZOI can help find the lowest concentration of an antimicrobial that fully stops bacterial growth. Clear, well-defined zones are needed for this.
- Drug discovery: With rising antimicrobial resistance, ZOI tests are vital for screening new compoundslike plant extracts or microbes that produce antimicrobial substances, to discover new treatments.
Limitations of Zone of Inhibition Test
- Only suitable for water-soluble antimicrobial agents โ This test method is not ideal for non-soluble antimicrobial agents. Meanwhile, water-soluble antimicrobial agents easily leach off from treated products and seep into agar medium, resulting in great activity against test microorganisms.
- A lack of the zone of inhibition does not mean that the product is ineffective โ The zone of inhibition test is a qualitative test method and has been questioned for its accuracy many times.
- Impact of Agar Composition on Antimicrobial Efficacy:Microbial growth agars themselves may interfere with the function of some antimicrobial agents.
Conclusion
At Microbe Investigation Switzerland, we understand the critical role of precise microbial testing in safeguarding health and ensuring product efficacy. Our ZOI, MIC and MBC testing services are designed to deliver accuracy and reliability, helping you meet regulatory requirements and exceed industry standards.
For detailed information on our ZOI testing services or to discuss how we can support your antibacterial research, please contact our expert team today.
Frequently Asked Questions
DR. Martinoz Scholtz
This test is a qualitative test method used to measure the antibacterial efficacy of products formulated to inhibit the growth of harmful bacteria.
This test is applicable to check the antibacterial efficacy of treated textiles, polymers, disinfectants, and antibiotics.
The turnaround time for this test is one week .
At Microbe Investigations Switzerland, we test using the following bacteria: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus hirae, Klebsiella pneumoniae, MRSA, Salmonella enterica ,Candida albicans.
It is determined by measuring the diameter, in millimeters, of the clear area around the test agent on the agar plate using a ruler or caliper.
The greater the zone of inhibition means, the greater is the antimicrobial potency, suggesting the test agent is effective in inhibiting microbial growth.
It indicates low antimicrobial efficacy, meaning that the test agent has less effectiveness against the test microorganism.
The size may also be affected by factors such as antimicrobial agent concentration and its diffusion rate, agar composition, incubation time, or the microbial strain used.
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