Systematic study on the evaluation method of surface antibacterial activity based on the fluorescent observation of bacterial growth

Abstract

Antibacterial and antiviral coating materials have attracted increasing attention for the prevention of infections caused by frequently touched surfaces in communities and hospitals. The standard assessment procedure for antibacterial surfaces involves bacterial culture on a film-covered substrate followed by transfer onto agar for colony counting (ISO22196:2011). However, this assessment lacks temporal and spatial information regarding bacterial growth, resulting in an incomplete and inaccurate evaluation of the antibacterial activity of the surface. In this study, we develop a novel evaluation procedure for antibacterial substrates that enables in situ visualization of bacterial growth on a surface with centimeter-scale spatial information using fluorescent protein-expressing bacterial cells and an image acquisition setup. The effects of equipment parameters on bacterial growth are systematically investigated to establish the standard evaluation conditions. Based on the optimized parameters, a quantitative evaluation of the antibacterial activity of the coating material is successfully demonstrated. The proposed method is expected to be useful in investigating the spatial and temporal distribution of bacterial growth on substrates.

Keywords: Antibacterial activity; Bacterial growth; Fluorescent observation; in situ visualization.

Fig. 1

Schematic of the experimental setup for the visualization of bacterial growth on a substrate. (a) Bacterial broth covered with an overhead transparent film on a glass substrate with four spacers to keep the distance between the substrate and the film. (b) Dark box to obtain fluorescent images using a digital camera under excitation by an LED light source.

Fig. 2

in situ visualization of GFP expressing E. coli growth using 4-cm square, 3.5-cm square, and 2-cm square overhead transparent films on substrates. The thickness of the spacers was 200 μm. Images acquired at 4, 6, 8, 24, and 48 h after bacterial inoculation on a substrate from left to right. Scale bars: 1 cm.

Fig. 3

Time course of the average fluorescent intensity from GFP expressing E. coli cultured on a substrate using overhead transparent films with a size of (a) 4-cm square, (b) 3.5-cm square and (c) 2-cm square. Black squares, red triangles, and blue circles indicate the results for the 200, 100, and 50 μm spacers, respectively (n = 3, mean ± standard deviation).

Fig. 4

Fluorescent intensity profiles along a line passing through overhead transparent films with a size of 4-cm square, 3.5-cm square, and 2-cm square. For each line, the fluorescent intensity was normalized to the maximum fluorescent intensity on the line and then plotted against the distance from the initial edge. The height of the spacers varied at 200, 100, and 50 μm. The red and black lines represent the results 24 and 48 h after inoculation, respectively.

Fig. 5

Coefficient of variation (CV) of fluorescent intensity on the substrate with different sizes of overhead transparent films. White bars indicate values obtained 24 h after inoculation and black bars indicate values obtained 48 h after inoculation (n = 9, means ± SD, **; p < 0.01, ****; p < 0.0001 in Turkey’s test following two-way ANOVA).

Fig. 6

Appearance of the substrates coated with white, nontransparent material (a) without (control) and (b) with antimicrobial agents (VK-500) evaluated in this study. (c) in situ visualization of GFP expressing E. coli growth on the control and VK-500 coated substrates. Images acquired at 4, 6, 8, 24, and 48 h (from left to right) after bacterial inoculation on the substrate. Scale bars: 1 cm. (d) Average fluorescent intensity analyzed from the obtained images (n = 3 at 0, 2, 4, 6, and 8 h, n = 9 at 24 and 48 h, means ± standard deviation). Black squares and red triangles indicate the results for coating materials without and with antibacterial agents, respectively. (e) Comparison of the average fluorescence intensity analyzed from the obtained images at 24 and 48 h after bacterial inoculation on the substrates (n = 9, means ± standard deviation, ****; p < 0.0001 in Student’s t-test).