Determination of minimum inhibitory and bactericidal concentrations of reduced graphene oxide–functionalized methylene blue for root canal disinfection: An in-vitro study

TL;DR

This study tests a new antimicrobial method using methylene blue combined with reduced graphene oxide to fight E. faecalis in root canals.

Contribution

The novel contribution is the functionalization of methylene blue with reduced graphene oxide for improved antibacterial efficacy under laser activation.

Findings

MB-rGO showed more consistent MIC and MBC values compared to MB alone.

Photoactivation of MB-rGO demonstrated potential as an enhanced antimicrobial agent against E. faecalis.

No significant statistical differences were found between MB and MB-rGO groups.

Abstract

Enterococcus faecalis (E. faecalis) is a persistent endodontic bacterium that often resists standard antimicrobial treatments. Improving the effectiveness of methylene blue (MB) by combining it with reduced graphene oxide (MB-rGO) and activating it using laser light presents a potential alternative antibacterial approach. Therefore, this study evaluated and compared the antimicrobial activity of laser-activated MB and MB-rGO against E. faecalis by determining their minimum inhibitory and bactericidal concentration (MIC and MBC).

This in-vitro study was performed for a period of eight months i.e from June 2024 to January 2025 at Universiti Sains Malaysia and King Faisal University Al-Ahsa Saudi Arabia. The antimicrobial activity of MB and MB-rGO against E. faecalis ATCC 29212 was assessed using the broth microdilution method following CLSI guidelines. A standardized inoculum (5 × 105 CFU/mL) was prepared in Mueller–Hinton broth. Stock solutions of MB and MB-rGO (800 µg/mL) were serially diluted to final concentrations ranging from 400 to 12.5 µg/mL. The test was conducted in 96-well microtiter plates, followed by photoactivation using a 660 nm diode laser for two minutes. Plates were incubated at 37°C for 24 hours. MIC was recorded as the lowest concentration without visible turbidity, while MBC was determined by subculturing from clear wells onto blood agar. Each test was performed three times independently under the same conditions to ensure reliability and consistency of the results.

Turbidity was observed at lower concentrations (12.5 and 25 µg/mL), while higher concentrations showed complete inhibition. Methylene blue (MB) demonstrated MIC and MBC values of 66.67 ± 28.87 µg/mL and 266.67 ± 115.47 µg/mL, respectively, whereas MB-rGO exhibited more consistent values of 50.00 ± 0.00 µg/mL and 200.00 ± 0.00 µg/mL. However, statistical analysis revealed no significant differences (p>0.05) between the groups.

Methylene blue functionalized with reduced graphene oxide (MB-rGO) showed more consistent antibacterial activity under photoactivation, suggesting its potential as an enhanced antimicrobial agent against E. faecalis.

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