Measurement of Antibacterial Activity on Plastics Surfaces and Other Non-Porous Surfaces

The ISO 22196 method is designed to quantitatively test the ability of plastics to inhibit the growth of microorganisms (Bacteriostatic) or kill them (Bactericidal), over a 24 hour period of contact. It is a relatively sensitive assay, meaning that it can detect low-level antimicrobial effects exerted over long periods of time. The second edition of the method extends its applicability to other Non-Porous Surfaces, no longer limiting it to only plastic surfaces. 

ISO 22196 was modeled after JIS Z 2801. The two methods are essentially the same.

Summary of the ISO 22196 Test
  • The test microorganism is prepared, usually by growth in a liquid culture medium. Per the method, two representative microorganisms are specified, S. aureus and E. coli
    • Depending on the Study Sponsor's testing objectives and products end goals, Microchem Laboratory can modify the test method to better fit your testing objectives, while maintaining a scientifically defensible study. This includes testing against more clinically and product-relevant microorganisms.
  • The suspension of the test microorganism is standardized by dilution in a nutritive broth (this affords microorganisms the potential to grow during the test).
  • Control and test surfaces are inoculated with microorganisms, in triplicate, and then the microbial inoculum is covered with a thin, sterile film. Covering the inoculum spreads it, prevents it from evaporating, and ensures close contact with the antimicrobial surface.
    • All microbiological assays run at Microchem Laboratory are performed with the necessary parallel controls to provide adequate comparisons at both the start of the test as well as after the contact time, in this case 24 hours. 
    • These controls allow us to fully evaluate the antimicrobial efficacy that can be attributed to the treated article's technology, and only this technology. This is achieved by including the proper controls, which control for any other variables that could affect the bacterial reduction being evaluated. 
  • Microbial concentrations are determined at "time zero" by elution followed by dilution and plating.
  • A control is run to verify that the neutralization/elution method effectively neutralizes the antimicrobial agent in the antimicrobial surface being tested.
  • Inoculated, covered control and antimicrobial test surfaces are allowed to incubate undisturbed in a humid environment for 24 hours.
  • After incubation, microbial concentrations are determined. The reduction of microorganisms relative to initial concentrations and the control surface is calculated.
    • By including the proper controls and being able to make these reduction calculations, this assay allows us to interpret whether the test substance is bacteriostatic, having the ability to inhibit the growth of microorganisms, or if the test substance is bactericidal, having the ability to kill them.

Strengths of the ISO 22196 Test
  • The method is quantitative and results tend to be reproducible, provided the inoculum does not spill off of the target area after being covered with the thin film.
  • The method tests for both bacteriostatic (growth-inhibiting) and bactericidal (bacteria-killing) properties.
  • Microbial concentrations are standardized, and bacteria are provided with nutrients during the incubation period, which provides them with ample opportunity to grow if surfaces aren't sufficiently antimicrobial. This is in contrast to certain other antimicrobial tests, where microbes are "incubated" in non-nutritive suspensions, which itself may be stressful over long periods.
  • The test method includes options to inoculation, such as the volume of the inoculum, to mediate for surfaces that do not lend themeselves to the normal testing parameters. This adds flexibility to the method and allows it to be used to evaluate very different surfaces with the same objective, to be antimicrobial. 
  • The method stipulates triplicate experimentation, which helps researchers estimate the precision of the individual tests and increases overall experimental accuracy.
  • The method includes a "pass/fail" criterion for the calculated levels of antimicrobial activity observed in test samples, making determinations of antimicrobial activity less discretionary.

Weaknesses of the ISO 22196 Test
  • The ISO 22196 method is not necessarily representative of actual surface contamination events, since a relatively dilute liquid microbial inoculum is spread over a considerable surface area, and then is kept wet (usually for a period of 24 hours). Most of the time, microbial contaminants dry quickly onto surfaces. This limits the time that an aqueous medium is available to facilitate interaction between the antimicrobial surface and microorganisms. This means that ISO 22196 is a "best-case" sort of test for many products.
    • This can be mediated by performing modifications to the method in order to provide a more aggressive challege. These include including the inoculum concentration as well as reducing the contact time. All of these things can be achieved through open communication between the Study Sponsor and the Scientist performing the study; a culture that is one of the lab's main focuses. 
    • All modifications are performed with the integrity and scientific reproducibility of the assay in mind. Any modifications deviating from the method are noted in the final report, in detail. 
  • Although the second edition of this method extends its applicability to other non-porous surfaces, it still excludes a number of surfaces that can be evaluated under this International Standard; these include building materials, textile products that have been treated to be non-porous, and photocatalytic materials. 

Though the ISO 22196 test is somewhat "best-case," it is an excellent way to quantify the antimicrobial activity level of an antimicrobial surface. Among the various tests for antimicrobial activity of surfaces, this has emerged as one of the industry standards.

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