ASSESSMENT OF ANTIMICROBIAL ACTIVITY OF HARD NON-POROUS SURFACES

The JIS Z 2801 method tests the ability of plastics, metals, ceramics and other antimicrobial surfaces to inhibit the growth
of microorganisms or kill them. The procedure is very sensitive to antimicrobial activity and has a number of real world
applications anywhere from the hospital/clinical environment to a household consumer company concerned with the
ability of a material they have to allow bacterial growth.

The JIS Z 2801 method is the most commonly chosen test and has become the industry standard for antimicrobial hard
surface performance in the United States. Below, you will find a summary of the JIS Z 2801 test method, along with some
of its strengths and weaknesses. The JIS Z 2801 test method method is designed to quantitatively test the ability of hard
surfaces to inhibit the growth of microorganisms or kill them, over a 24 hour period of contact.

The JIS Z 2801 procedure has been adopted as an International Organization for Standardization (ISO) procedure, ISO
22196.

SUMMARY OF THE JIS Z 2801 TEST

• The test microorganism is prepared, usually by growth in a liquid culture medium.

• The suspension of 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.
• 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.

STRENGTHS OF THE JIS Z 2801 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 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 JIS Z 2801 TEST

• The JIS Z 2801 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 JIS Z 2801 is a "best-case" sort of test for many products.
• It is not uncommon for "control" plastics to exert low-level antimicrobial activity, so identification of an ideal
control surface may be challenging.

Though the JIS Z 2801 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.
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.
o 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.
o 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.
o 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.
o 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.
o 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.
o 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.