Er:YAG Laser Energy Optimization for Reducing Single-Species Microbial Growth on Agar Surfaces In Vitro

Abstract

Background: Standardized Er:YAG laser settings for microbial reduction remain undefined, and existing studies rarely compare multiple species under identical conditions. This work aimed to characterize susceptibility across selected microorganisms using a controlled agar-based surface growth model.

Methods: Six reference strains (E. coli, S. aureus MSSA, S. aureus MRSA, E. faecalis, P. aeruginosa, and C. albicans) were cultured on agar and exposed to Er:YAG irradiation. Two experimental phases were conducted: (1) inhibition zone mapping using energies between 30 and 400 mJ at 1 Hz, with tapered and flat laser tips; and (2) quantification of viable surface coverage after irradiating mature 96 h cultures with 80, 130, 180, and 230 mJ at 10 Hz in contact mode. ImageJ analysis was used to measure inhibition diameters and remaining coverage. Data were evaluated using two-way ANOVA.

Results: All microorganisms showed measurable inhibition at every tested energy level, with diameter increasing proportionally to energy. E. coli and E. faecalis produced the largest inhibition zones in the mapping phase, while P. aeruginosa and C. albicans required higher energies to reach comparable levels. Mature surface cultures showed progressive reductions in viable coverage; the strongest effects occurred at 230 mJ. The tapered tip generated broader inhibition zones at lower energies compared with the flat tip.

Conclusions: Er:YAG laser irradiation produces consistent, energy-dependent antimicrobial effects on single-species agar-based surface growth, with clear differences in species susceptibility and tip performance. The identified parameter ranges provide a quantitative foundation for future in vitro studies aiming to refine Er:YAG-based microbial reduction strategies.

Keywords: Candida albicans; Enterococcus faecalis; Escherichia coli; Pseudomonas aeruginosa; Staphylococcus aureus; agar; in vitro; laser therapy; lasers.

Figure 1

(a) Template Design for Er:YAG Laser Irradiation Showing Energy Distribution Across 32 Target Points. (b) The experimental setup involved a Petri dish filled with agar. A 100 µL suspension was applied onto a template featuring 32 marked points, each representing a target site for Er:YAG laser exposure (AdverEvo, Morita, Osaka, Japan).

Figure 2

Schematic of the irradiated grid showing the selected squares on the agar plates. The top left square was irradiated with 80 mJ, the top right with 130 mJ, the bottom left with 180 mJ, and the bottom right with 230 mJ.

Figure 3

(a) Agar plate showing the four predefined square irradiation zones (12 × 12 mm) after laser application. (b) Sampling using Rodac IRR LAB-Agar contact plates (c) Schematic representation of the irradiation pattern within a single square over time. At 0 s the area is untreated. At 60 s the laser tip has passed over the surface in one diagonal direction, at 120 s a second diagonal pass is added to create a crosshatch pattern, and at 180 s vertical passes are added.

Figure 4

Comparative analysis of the antimicrobial activity of Er:YAG laser irradiation using two different laser tips. (a) Growth inhibition zone diameters (mm) for six single-species cultures, Pseudomonas aeruginosa (PA 27853), Staphylococcus aureus (SA 25923 and SA 43300), Enterococcus faecalis (EF 29212), Escherichia coli (EC 25922), and Candida albicans (CA 10231), irradiated with a tapered tip across increasing energy levels (30–400 mJ). (b) Corresponding results obtained using a flat tip under identical conditions.

Figure 5

Er:YAG laser irradiation patterns obtained using two different fiber tip types. (a) Inhibition zones produced with the tapered tip (b) Inhibition zones generated with the flat tip under identical irradiation parameters.

Figure 6

The experimental procedure used to quantify the percentage of surface area covered by single-species culture following Er:YAG laser irradiation. (a) Visible microbial growth within the marked areas indicates residual culture following treatment. (b) Processed image generated in ImageJ-Fiji software used for quantitative analysis. The red overlay represents the area covered by residual microbial colonies. This method allowed objective calculation of the percentage of coverage relative to the total irradiated surface for each tested microorganism.

Figure 7

Effect of Er:YAG laser energy on the mean percentage surface coverage of viable coverage for six single-species cultures. The bar chart shows the mean (±SD) percentage of surface area covered by surviving colonies after laser irradiation at four different energy levels (80, 130, 180, and 230 mJ). Error bars represent the standard deviation. (1) Escherichia coli ATCC 25922 (2) Staphylococcus aureus ATCC 25923 (MSSA) (3) Staphylococcus aureus ATCC 43300 (MRSA) (4) Enterococcus faecalis ATCC 29212 (5) Pseudomonas aeruginosa ATCC 27853 (6) Candida albicans ATCC 10231.