Detection of Biofilm Formation on Material Surfaces by Ag+ Coating
Authors: Kogo Takeshi , Sugi Kazufumi , Kanematsu Hideyuki , Kai Hotaka , Ogawa Akiko , Hirai Nobumitsu , Takahashi Toshiyuki , Kato Takehito 
Published: July 2022

The evaluation of biofilm formation is important, given the ubiquity and problematic nature of biofilms in industrial and medical settings, as well as in everyday life. Basically, biofilms are formed on substrates. Therefore, it is essential to consider the properties of the substrates during biofilm evaluation. The common dye staining method to evaluate biofilm formation requires a short evaluation time and enables the evaluation of a large area of the sample. Furthermore, it can be easily determined visually, and quantitative evaluation is possible by quantifying color adsorption. Meanwhile, the dye staining method has the problem of adsorption even on substrate surfaces where no biofilm has formed. Therefore, in this study, we focused on Ag+ reduction reaction to devise a novel biofilm evaluation method. Ag+ is highly reductive and selectively reacts with organic substances, such as saccharides, aldehydes, and proteins contained in biofilms, depositing as metallic Ag. First, to simply evaluate biofilm formation, we used a glass substrate as a smooth, transparent, and versatile oxide material. We observed that the amount of Ag deposited on the substrate was increased proportionally to the amount of biofilm formed under light irradiation. Upon comparing the Ag deposition behavior and adsorption behavior of crystal violet, we discovered that for short immersion times in AgNO3 solution, Ag deposition was insufficient to evaluate the amount of biofilm formation. This result suggests that the Ag reduction reaction is more insensitive than the crystal violet adsorption behavior. The results of the Ag deposition reaction for 24 h showed a similar trend to the crystal violet dye adsorption behavior. However, quantitative biofilm evaluation using the proposed method was difficult because of the Ag+ exchange with the alkali metal ions contained in the glass substrate. We addressed this issue by using the basic solution obtained by adding an ammonia solution to aqueous AgNO3. This can cause Ag+ to selectively react with the biofilm, thus enabling a more accurate quantitative evaluation. The optimum was determined at a ratio of distilled water to aqueous ammonia solution of 97:3 by weight. This biofilm was also evaluated for materials other than ceramics (glass substrate): organic material (polyethylene) and metal material (pure iron). In the case of polyethylene, a suitable response and evaluation of biofilm formation was successfully achieved using this method. Meanwhile, in the case of pure iron, a significantly large lumpy deposit of Ag was observed. The likely reason is that Ag precipitation occurred along with the elution of iron ions because of the difference in ionization tendency. It could be concluded that the detection of biofilm formation using this method was effective to evaluate biofilm formation on materials, in which the reduction reaction of [Ag(NH3)2]+ does not occur. Thus, a simple and relatively quantitative evaluation of biofilms formed on substrates is possible using this method.