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Zone of Inhibition Test

Measures the Resistance of Pathogenic Bacteria to Antibacterial agents

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Zone of Inhibition Test

Zone of Inhibition test (also referred to as Kirby-Bauer test) is used to determine the susceptibility or resistance of pathogenic bacteria to antibacterial agents.

In comparison to other laboratory test methods, the zone of Inhibition test is an inexpensive and fast way to determine antibacterial activity. 

Major advantage is this test can be used for a variety of antibacterial product samples such as treated textiles, polymers, disinfectants, and antibiotics.

Zone of Inhibition Test Requirement


  • Mueller-Hinton agar plates
  • Sterile swabs and forceps
  • Pure bacterial culture
  • Sample of antibacterial product (size of 6 mm)

Zone of Inhibition Test Method

  1. A swab of pure bacterial culture is evenly spread over Mueller-Hinton agar plates. 
  2. Using a sterile forcep, the treated product sample is placed on the media plate.
  3. This petri plate is kept for incubation for 18-24 hours at 36°C along with other optimal conditions for bacterial growth.
  4. After the incubation period, a clear area (zone of inhibition) around the antibacterial product sample is observed and measured.
  5. Treated products with strong antibacterial activity form a larger zone of inhibition or vice versa.
Zone of Inhibition Test

Limitations of Zone of Inhibition Test

  • Only suitable for water-soluble antimicrobial agents – This test method is not suitable for non-soluble antimicrobial agents. While, 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 – The zone of inhibition test is a qualitative test method and has been questioned for its accuracy many times.

Based on the observations, it is concluded that growth media can also hinder the growth of microorganisms instead of antimicrobial agents.

Difference between Minimum Inhibitory Concentration (MIC) and Zone of Inhibition (ZOI)

Minimum Inhibitory Concentration (MIC) – It is the lowest concentration of antimicrobial agent that prevents the visible growth of microorganisms after incubation. The Minimum Inhibitory Concentration test determines the antimicrobial activity of a test agent against a specific bacteria.

Zone of Inhibition (ZOI) – It is a clear circular area around antimicrobial discs in which bacteria are unable to grow.

Both these terms are inversely proportional to each other.  It depends on the susceptibility of the test organism to the antimicrobial agent.  Higher the susceptibility, larger the zone of inhibition and lower the MIC.  Whereas, a higher MIC value and a small zone of inhibition indicate that bacteria are resistant to antimicrobial agents.

When it comes to evaluating the antimicrobial activity of treated products, quantitative tests are found quite useful as they produce more accurate and clear results. 

At MIS (Microbe Investigations), we use international standards such as ISO 22196, JIS L 1902ISO 20743, and JIS Z 2801 to evaluate the antibacterial activity of treated products.

We offer a complete spectrum of antimicrobial testing services for all major industries (Textiles, Plastics, Coatings, and Surface disinfectants) developing antimicrobial products. In our well-equipped laboratories, we determine the antimicrobial effectiveness of products using international standards such as ISO, JIS, AATCC, and ASTM.

To get a quote and a free expert consultation, contact here. With a customer-centric approach, our clear focus is on ensuring on-time delivery with accuracy.


Zone of Inhibition test is a qualitative test method used to measure the antibacterial efficacy of products formulated to inhibit the growth of harmful bacteria.

Zone of Inhibition test is applicable to check the antibacterial efficacy of treated textiles, polymers, disinfectants, and antibiotics. 

The Zone of Inhibition test takes 3-4 days to complete.

At Microbe Investigations, we test for Zone of Inhibition using the following bacteria: Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538P), Enterococcus hirae (ATCC 10541), Klebsiella pneumoniae (ATCC 4352), MRSA (ATCC 33591), Salmonella enterica ( ATCC 10708), Candida Albicans (MTCC 3017). Additional strains can be added on request.

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