ADVANCED MATERIALS & PROCESSES | JANUARY/FEBRUARY 2024 26 materials used in products—and their surfaces—and has investigated biofilm formation and growth as one of the material’s characteristics. SIAA organized a committee and several subcommittees to carefully study this issue, and after several preliminary votes and discussions, a new ISO standard (ISO 4768:2023) was successfully adopted by a final vote on July 18, 2023. BIOFILM EVALUATION PROCESS The authors determined that the most effective method for biofilm evaluation is staining, which is practical, intuitive, easy to perform, and inexpensive. Various staining procedures are possible. Biofilms are mainly composed of bacteria, EPS, and water, and many reagents have been developed to stain bacteria in biofilms. However, as discussed previously, the essence of a biofilm is EPS. Therefore, creating a staining agent that can stain EPS, bacteria, and other components of biofilms in the broadest time range is important. From this perspective, crystal violet is the most favorable. Crystal violet is a stain with a triphenylmethane backbone, which is also used as a pH indicator and in Gram stains for dyeing bacteria. The cations with triphenylmethane groups are ionized to chloride ions in aqueous solution. Therefore, the crystal violet is electrically attracted to polarizable polymers, where it adsorbs and develops color. Crystal violet is ideal because it can stain the entire biofilm. Figure 2 shows the biofilm formation process according to the new standard (ISO 4768:2023) as follows: Prepare a 4-cm glass plate; place a 3-cm square specimen of the material to be tested onto the glass plate and attach it with double-sided tape; then place specimen in a polyethylene container with a dilution of 103 CFU/ml of Staphy- lococcus epidermidis precultured in 1/5 TSB medium. After 48 hours of immersion, specimens are removed, rinsed with sterile water, and stained with 0.1% crystal violet for 30 minutes. The specimen is then rinsed with sterile water again, wiped with a nonwoven cloth moistened with alcohol to remove the crystal violet, and immersed in 1% sodium dodecyl sulfate to extract the purple-stained biofilm. The biofilm is then irradiated with light at 590 nm and the absorbance is measured. The absorbance is quantitatively related to the biofilm on the material surface. However, the problem is that a higher absorbance value indicates a higher amount of biofilm. Although biofilms can be beneficial in some cases, they typically tend to be harmful to human health and negatively impact the products they attach to. Therefore, the index should be set so that the higher the value, the less likely a biofilm will form. In addition, the absolute value of absorbance varies depending on the device, production lot, and environment, and using correlation values is more practical than absolute values. For this reason, an index of anti-biofilm activity, R, was proposed in the new ISO standard, and is shown in equation (1). R = {( A0 - A1)/ A0} x 100 (%) (1) where A0 = absorbance of control specimen; and A1 = absorbance of target specimen. This formula allows for relative evaluation and eliminates many of the drawbacks of using absorbance values. FUTURE CONSIDERATIONS SIAA is actively considering the possibility of certifying products based on this new international standard, using a system that should be complete by mid-2024. In the meantime, the authors plan to introduce their ideas on using the standard from the perspectives of materials science, materials engineering, and especially materials surface engineering. Figure 3 shows a schematic of potential antibacterial materials development. In this example, the base Fig. 2 — Biofilm formation according to new ISO standard, ISO 4768:2023. Fig. 3 — Application of ISO standard to development of new anti-biofilm materials. If the difference between samples in the anti-biofilm index is greater than 20%, this would indicate that the anti-biofilm properties are approaching the target value for the new material.
RkJQdWJsaXNoZXIy MTYyMzk3NQ==