Zone of Inhibition Test for Antimicrobial Activity

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Zone of Inhibition Test, also called a Kirby-Bauer Test.

This is a qualitative method used clinically to measure antibiotic resistance and industrially to test the ability of solids and textiles to inhibit microbial growth.

Researchers who develop antimicrobial textiles, surfaces, and liquids use this test as a quick and easy way to measure and compare levels of inhibitory activity.

With this method, approximately one million cells from a single strain are spread over an agar plate using a sterile swab, then incubated in the presence of the antimicrobial object (ex: an oxacillin disk, pictured below). If the bacterial or fungal strain is susceptible to the antimicrobial agent, then a zone of inhibition appears on the agar plate, such as on the agar plate on the left-hand side of the photo below. If it is resistant to the antimicrobial agent, then no zone is evident, such as on the agar plate on the right-hand side of the photo below.

Summary of the Zone of Inhibition Test

Photograph: Left Plate is of S. aureus with Oxacillin disk, Right Image is lawn growth of S. aureus.

  • A bacterial or fungal strain of interest is grown in pure culture.
  • Using a sterile swab, a suspension of the pure culture is spread evenly over the face of a sterile agar plate.
  • The antimicrobial agent is applied to the center of the agar plate (in a fashion such that the antimicrobial doesn’t spread out from the center). A hole can be bored in the center of an agar for a liquid substance.
  • The agar plate is incubated for 18-24 hours (or longer if necessary), at a temperature suitable for the test microorganism.
  • If antimicrobial agent leaches from the object into the agar and then exerts a growth-inhibiting effect, then a clear zone (the zone of inhibition) appears around the test product.
  • The size of the zone of inhibition is usually related to the level of antimicrobial activity present in the sample or product – a larger zone of inhibition usually means that the antimicrobial is more potent.

Strengths of Zone of Inhibition Testing

  • Zone of inhibition testing is fast and inexpensive relative to other laboratory tests for antimicrobial activity.
  • Zone of inhibition testing is especially well suited for determining (albeit qualitatively) the ability of water-soluble antimicrobials to inhibit the growth of microorganisms.
  • A number of samples can be screened for antimicrobial properties quickly using this test method.
  • A variety of antimicrobial product types can be tested using this method. Liquids, coated antimicrobial surfaces, and antimicrobial-impregnated solid products can all be tested for their ability to produce a zone of inhibition.

Weaknesses of Zone of Inhibition Testing

  • Antimicrobial agents that leach out of the object and into the aqueous agar matrix, such as silver ions, usually show better results than antimicrobials that stay affixed to the object or textile or that are not water-soluble.
  • Zone of Inhibition tests do not necessarily indicate that microorganisms have been killed by an antimicrobial product – just that they have been prevented from growing.
  • Microbial growth agars themselves may interfere with the function of some antimicrobial agents.
  • The method cannot be used to test the activity of antimicrobial agents against viruses, since viruses don’t “grow” on agar plates like bacteria (viruses don’t replicate outside of their host organisms).
  • The method has some natural variability, and zones of microbial inhibition do not always have clear or regular boundaries.
  • The method is not classically quantitative (though sometimes the diameter of the zones of inhibition are measured and recorded).

Zone of Inhibition Testing is a fast, qualitative means to measure the ability of an antimicrobial agent to inhibit the growth of microorganisms. In the world of antimicrobial substances/surfaces, the degree to which these materials are inhibitory can be of vital importance to the health of the consumer. This test is an outstanding qualitative way for manufacturers of antimicrobial surfaces/substances to be able to compare the inhibition levels of their products.

Microchem Laboratory was built around antimicrobial innovation and antimicrobial research and development. If your company is interested in screening chemicals, products, or antimicrobial treatments for their ability to inhibit the growth of microorganisms, the zone of inhibition test may be a great place to start.

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