An overview of methods used to evaluate the efficacy of antibacterial treated surfaces and textiles
by Vivienne Mahon PhD a Senior Researcher at airmid healthgroup
In general, rather than being free living, most microorganisms exist attached onto surfaces. These microorganisms then have the potential to transfer to other locations e.g if they come into contact with human skin or food items. If microorganisms are transferred to an environment that has favourable conditions for growth, they may multiply and eventually cause adverse effects in exposed individuals. An antimicrobial treatment could prevent a surface from becoming a reservoir of infection.
Therefore antimicrobial surfaces are of significant interest to both consumers and manufacturers, and this is an area that has been vigorously marketed and pursued. A number of different antimicrobial agents have been immobilized onto a wide range of both non-porous surfaces (such as plastics and metals) and porous surfaces (such as natural and synthetic fabrics).
It is important that antimicrobial surfaces have undergone testing to confirm their efficacy and to provide scientific evidence to support any claims that the manufacturer wishes to make about them. The test method that is used should be reproducible and careful consideration should be given to crucial parameters such as experimental conditions, the use of appropriate controls and the type and concentration of microorganisms used. Indeed the type of microorganisms used in the test will affect the claims that can be made about the surface e.g. whether it is antibacterial, antifungal or antiviral. A number of standard testing methods for the assessment of antimicrobial surfaces have been developed by bodies such as the International Organization for Standardization (ISO), the American Society for Testing and Materials (ASTM) and the American Association of Textile Chemists and Colorists (AATCC). In this article standard methods that use bacteria for the test organisms will be discussed, although there have been reports of some of the methods described below being modified to investigate antifungal and/or antiviral effects.
For non-porous (e.g. plastic, metal, ceramic) surfaces the Japanese Industrial Standard JIS Z 2801 is the test method most frequently used by manufacturers. This widely recognised method was drawn up by a consortium of research bodies, universities and manufacturers of antibacterial agents. The JIS Z 2801 standard served as the basis for ISO 22196, the international standard for the measurement of antibacterial activity on non-porous surfaces, and the two methods are essentially the same. Both involve placing a droplet of a suspension of either Escherichia coli or Staphylococcus aureus directly onto the surface being tested. A coverslip is then placed on top of the droplet, thus spreading the bacterial suspension and keeping it in intimate contact with the test surface. After a 24 hour incubation at 35°C the bacterial suspension is released from between the coverslip-test sample sandwich and the number of viable bacterial cells that have survived is determined. Some of the strengths of the JIS Z 2801/ISO 22196 method are that it is (i) quantitative, (ii) carried out in triplicate and (iii) said to have good reproducibility. However there are also some drawbacks to this test. The experimental conditions are more of a best-case scenario for the antimicrobial surface being tested rather than a reflection of the conditions that are likely to occur in a real life situation. In addition it is possible that the results might be affected by bacteria adhering to the coverslip. This has led some researchers to modify the test method by omitting the coverslip.
The ASTM E2149 standard method for the assessment of the antimicrobial efficacy of immobilized antimicrobials is frequently used to test both non-porous and porous materials. Also known as the “dynamic shake flask” test, the ASTM E2149 method involves the immersion of the test sample in a flask containing a known concentration of bacteria (typically Klebsiella pneumonia). The flask is mechanically agitated for a specified time period to ensure good contact between the bacteria and the test sample after which the population of viable bacteria that remains is enumerated. The antimicrobial efficacy is determined by comparing the number of organisms present before and after exposure to the test item, or by comparing the performance of the treated test item to that of an untreated negative control item. Once again a major drawback of this approach is that the test does not replicate the situations under which the test item would be used, and a positive result under this method may not be reproducible in a test simulating more real-life conditions. Despite this shortcoming, the strength of ASTM E2149 is that it can be used to test irregular shaped objects; however testing according to JIS Z 2801/ISO 22196 is preferable for flat surfaces.
For porous surfaces such as textiles, the main form of testing is typified by the AATCC Test Method 100. A bacterial suspension is completely absorbed into swatches of the test textile so that the bacteria are in intimate contact with the treated surface. After an incubation period in a high humidity environment, any surviving organisms are recovered from the swatches in a neutralising solution (broth). The number of bacteria recovered from the test textile is compared to the number of bacteria initially present or to the number present after incubation with an untreated control textile (if available). While this method is considered the textile industry standard, it has some weaknesses: a full nutrient broth is used during testing, which enables more vigorous bacterial growth than would be expected in most real-world environments, the test is usually only carried out once as there is no stated requirement for repeated replicates to be performed, and the success criteria of the test is vague and open to interpretation.
The Japanese testing method for antibacterial activity of textiles, JIS L 1902, includes a quantitative test that is similar to some aspects of AATCC 100 but it is carried out in triplicate and uses a diluted broth with a lower level of nutrients. JIS L 1902 also contains a qualitative test (modelled on AATCC test method 147) in which replicate textile samples are placed on the surface of agar plates that have been coated with a bacterial culture to determine if bacterial growth around the textile is inhibited. The ISO 20743 method is largely modelled on JIS L 1902 and includes a variant of AATCC 100. JIS L 1902 and ISO 20743 are said to be the most universal of the bacterial efficacy tests for textile applications.
As can be seen from above, there are a range of standardised methods available for testing antibacterial surfaces and textiles. It is certainly worthwhile to use these “off-the-shelf” methods as a first step to demonstrate proof of principle, i.e. to determine the efficacy of the incorporated antimicrobial in the treated surface/textile against a target organism. However it is worthwhile proceeding to further testing to fully explore the potential activity of the treated article. This could be done either by modifying the standard testing methods or by designing bespoke testing, from simulating realistic exposure conditions in the lab through to in-use evaluation (i.e. field studies). It is only then that issues arising in real life situations can be assessed. These issues include the effect of repeated fouling and cleaning of the antimicrobial surface/textile and the activity of the antimicrobial over long periods of time.
Contact Graeme Tarbox if you have any questions on the issues raised in this article and to learn how we can add value to your company: gtarbox(at)airmidhealthgroup(dot)com
1 Verran, J. (2002) Biofouling in Food Processing: Biofilm or Biotransfer Potential? Food and Bioproducts Processing 80 (4), 292-298
2 OECD. (2007) Analysis and assessment of current protocols to develop harmonised test methods and relevant performance standards for efficacy testing of treated articles/treated materials. ENV/JM/MONO(2007)4 (PDF 283 KB)
3 Haldar, J. et al. (2007) Preparation, application and testing of permanent antibacterial and antiviral coatings. Nature Protocols 2 (10), 2412-2417
4 Green, J.B. et al. (2011) Review of immobilized antimicrobial agents and methods for testing. Biointerphases 6 (4), CL2-43
5 JIS Z 2801:2000 Antimicrobial Products – Test for Antimicrobial Activity and Efficacy. Japanese Standards Association, Tokyo, Japan
6 Vreuls, C. et al. (2010) Biomolecules in multilayer film for antimicrobial and easy-cleaning stainless steel surface applications. Biofouling 26 (6), 645-656
7 ISO 22196:2011 Measurement of antibacterial activity on plastics and other non-porous surfaces. International Organization for Standardization, Geneva, Switzerland
8 ASTM E2149-13a Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions. American Society for Testing and Materials
9 AATCC Test Method 100, Antibacterial Finishes on Textile Materials: Assessment of. Technical Manual of the American Association of Textile Chemists and Colorists
10 Swofford, H.W. (2010) An Overview of Antimicrobial Testing for Textile Applications. AATCC Review November/December, 51-55 (PDF 234 KB)
11 JIS L 1902:2015 Textiles - Determination of antibacterial activity and efficacy of textile products. Japanese Standards Association, Tokyo, Japan
12 AATCC. Test Method 147, Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method. Technical Manual of the American Association of Textile Chemists and Colorists
13 ISO 20743:2013 Textiles - Determination of antibacterial activity of antibacterial finished products. International Organization for Standardization, Geneva, Switzerland