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Microbial Growth in Dental Unit Waterlines and Biofluorescence Monitoring Using QLF Technology After Regular Disinfection
J Dent Hyg Sci 2024;24:327-34
Published online December 31, 2024;  https://doi.org/10.17135/jdhs.2024.24.4.327
© 2024 Korean Society of Dental Hygiene Science.

Seong-Chan Park1 , Ji-Hyun Min2 , and Sun-Young Han1,3,†

1Department of Dental Hygiene, Yonsei University Graduate School, Wonju 26493, 2Department of Dental Hygiene, College of Health and Medical Sciences, Cheongju University, Cheongju 28503, 3Department of Dental Hygiene, College of Software and Digital Healthcare Convergence, Yonsei University, Wonju 26493, Korea
Correspondence to: Sun-Young Han, https://orcid.org/0000-0001-7578-782X
Department of Dental Hygiene, College of Software and Digital Healthcare Convergence, Yonsei University, 1 Yeonsedae-gil, Heungeop-myeon, Wonju 26493, Korea
Tel: +82-33-760-5562, Fax: +82-33-760-5575, E-mail: syhan0724@yonsei.ac.kr
Received November 28, 2024; Revised December 8, 2024; Accepted December 10, 2024.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Dental unit waterlines (DUWLs) are critical components of dental care. However, their small diameter and extended water stagnation time make them prone to microbial contamination and biofilm formation. This study evaluated microbial contamination following routine DUWL disinfection and investigated the applicability of quantitative light-induced fluorescence (QLF) technology as a device for monitoring changes in biofluorescence within DUWLs.
Methods: This study evaluated microbial growth in DUWLs after routine disinfection with hydrogen peroxide every two weeks in a dental hygiene training laboratory. Water samples were collected from three devices (3-way syringes, ultrasonic scalers [US], and cup fillers) immediately after disinfection (baseline) and two weeks later. Colony-forming units (CFUs) were cultured on R2A agar and quantified. QLF technology, specifically QLF-Digital (QLF-D), was employed to measure biofluorescence intensity and contamination levels. Statistical analyses included the Kruskal-Wallis and paired t-tests.
Results: Microbial counts met the contamination standards (<100 CFU/ml) immediately after disinfection but increased significantly after two weeks. Among these devices, the US exhibited the highest contamination and biofluorescence levels, highlighting their vulnerability to microbial growth. The red fluorescence intensity of the microbial pellets also increased significantly over two weeks, correlating with the microbial contamination levels.
Conclusion: These findings suggest that although regular disinfection effectively reduces initial contamination, its efficacy diminishes over time, especially for high-risk devices. Furthermore, QLF-D is useful for monitoring microbial growth and biofilm formation, indicating its potential for routine DUWL contamination assessments. Enhanced protocols are recommended to improve the long-term waterline quality for high-risk devices.
Keywords : Biofilms, Biofluorescence monitoring, Dental unit waterlines, Disinfection, Quantitative light-induced fluorescence
Introduction

1. Background

Dental unit waterlines (DUWLs) are critical components in dental procedures that supply water to various devices such as high-speed handpieces, low-speed handpieces, 3-way syringe (3W), ultrasonic scalers (US), and cup fillers (CF). For these devices to function properly, the dental units must be connected to water supply systems, drainage systems, and air compressors. Each device receives the necessary water and air supply through these connections.

Medical devices and piping systems with fluid flows create environments conducive to microbial colonization. Biofilms are well-organized communities of microorganisms, including bacteria, fungi, protozoans, and diatoms1). Biofilms can form on the tubes, waterlines, and handpieces of DUWLs, posing a significant risk of cross-contamination and infection transmission in dental settings owing to the pathogenic microorganisms they harbor2). Water discharged from DUWLs can act as a medium for microbial transmission, potentially contaminating the oral cavities of patients and leading to severe infections3). Healthcare professionals and patients can inhale aerosols generated during dental procedures containing water droplets from DUWLs. This risk is particularly concerning for immuno-compromised and older individuals, who are more susceptible to infections2,4,5). The Centers for Disease Control and Prevention (CDC) highlighted the increased risk of bacterial contamination in DUWLs due to water stagnation, emphasizing the importance of regular disinfection, water drainage, use of water filters, and routine water quality monitoring6). These measures are recommended to mitigate microbial growth and ensure the safety of the dental care environment.

Various methods are used in dental clinics to control or prevent biofilm formation in DUWLs7,8). Common approaches include antimicrobial agents and adherence to routine cleaning and disinfection protocols9). However, pathogenic microorganisms embedded within biofilms often exhibit significant resistance to antimicrobial treatments, leading to persistent contamination despite regular disinfection efforts.

Additionally, factors such as the clinical environment, the nature of dental procedures, the aging of municipal water systems supplying the dental unit, and the adequacy of water drainage practices can influence the rate of pathogenic biofilm growth. Therefore, it is essential to monitor contamination levels in each clinical or training setting and adjust disinfection intervals accordingly.

Quantitative light-induced fluorescence (QLF) technology, which detects red fluorescence emitted by mature biofilms, is widely used in dental clinics to identify dental plaques, dental caries, and bacteria-related oral diseases at an early stage. By leveraging QLF’s ability to detect biofilm maturity, the contamination levels of DUWLs can be monitored. This application would enhance infection control in dental settings and expand the clinical utility of QLF technology.

2. Objectives

This study aimed to investigate the increase in microbial contamination associated with biofilms by collecting and culturing microorganisms from dental unit immediately after disinfection and two weeks later in a dental hygiene laboratory that adheres to the disinfection intervals recommended by dental unit suppliers. By observing the extent of microbial growth, this study sought to evaluate the potential of microbial fluorescence to assess waterline contamination and monitor water quality.

Materials and Methods

1. Study subjects

This study was conducted in the dental hygiene laboratory at University A, where regular waterline disinfection was performed every two weeks using 3% hydrogen peroxide (H2O2). H2O2, recommended by the manufacturer as a disinfectant, was used for waterline disinfection by diluting distilled water with 35% H2O2 (hydrogen peroxide; Green Pharmaceutical Co., Ltd., Seoul, Korea) to prepare a final concentration of approximately 3% H2O2. In addition to routine waterline disinfection, water flushing of the 3W and US was performed 1∼2 minutes before and after each practice session. Samples were collected from randomly selected dental unit among those newly installed in 2021. The sampling sites included the 3W, US, and CF. To observe the growth of microbial contamination, samples were collected at two time points: (1) within one hour after disinfection (baseline) and (2) two weeks later, just before the next scheduled disinfection. The tip of the 3W was removed to minimize contamination during sample collection, and the US body was detached. The surrounding areas of the sample outlets were disinfected with 70% alcohol, and 30 ml water samples were collected in 50 ml conical tubes (Conical Tube; SPL Life Sciences, Pocheon, Korea)10). All samples were maintained at 4°C and analyzed immediately upon arrival at the laboratory.

2. Evaluation of water quality contamination via bacterial culturing

Bacterial culturing was conducted using an R2A agar medium (KisanBio Co., Ltd., Seoul, Korea), as recommended by the Standard Methods for Drinking Water Quality Testing11). The collected samples were tested for cell counting in three forms: undiluted, 10-fold diluted, and 100-fold diluted. Before plating, the samples were thoroughly vortexed to ensure homogeneity12). For each sample from the 3W, US, and CF, 100 µl was plated in triplicate onto agar plates and spread using a sterilized glass spreader. The plates were then incubated under aerobic conditions at 37°C for 72 hours13). After incubation, the colony-forming units per unit volume (CFU/ml) were counted, and the median value for each sample was calculated. This process was conducted twice, corresponding to two sampling points, at baseline and two weeks later. The degree of water contamination was evaluated based on a threshold of 100 CFU/ml, as suggested in the Standard Policy Manual for Dental Infection Control by the Ministry of Health and Welfare14).

3. Comparison of colony biofluorescence by sampling time

To investigate the differences in microbial biofluorescence between samples collected immediately after disinfection and after two weeks of use, colonies cultured for water quality assessment were photographed using QLF-Digital (QLF-D; Inspektor Research Systems B.V., Amsterdam, Netherlands). The imaging conditions were set as follows: shutter speed=1/4 s, aperture value=f/8.0, and International Organization for Standardization (ISO) speed=1,600. QLF-D imaging was performed immediately after the water quality assessment, with two sessions conducted in total, corresponding to two sampling time points: baseline and two weeks later.

4. Observation and analysis of pellet fluorescence

To compare the redness intensity caused by all the microorganisms present in the samples, pellets were formed and photographed using QLF-D. Immediately after sample collection, 3 ml of each sample was added to 7 ml of R2A broth medium (KisanBio Co., Ltd.) in 15 ml conical tubes, followed by incubation at 37°C under aerobic conditions for 72 hours. After incubation, the samples were vortexed and divided into 1 ml portions in microcentrifuge tubes (Neurex Life Science, Seoul, Korea). The samples were centrifuged at 13,500 rpm for 5 minutes (Centrifuge CF-10; Daihan Scientific Co., Ltd., Gangwon, Korea) to form pellets. The supernatant was removed using a micropipette, and the pellets were photographed using a QLF-D under the same imaging conditions used for colony fluorescence observation (shutter speed=1/4 s, aperture value=f/8.0, ISO speed=1,600). This process was conducted twice, corresponding to two sampling time points: baseline and two weeks later. QLF-D images were analyzed using Adobe Photoshop (Adobe Photoshop 2022; Adobe, San Jose, CA, USA). Three arbitrary locations on each pellet were selected to extract RGB values, and the average red value (R-value) was used for analysis. To assess the change in red value after two weeks of dental unit use, the difference in R-values between the two time points (ΔR) was calculated.

5. Statistical analysis

All statistical analyses were performed using the IBM SPSS Statistics software (version 29.0; IBM Corp., Armonk, NY, USA). Normality was assessed using the Shapiro-Wilk test. Group comparisons of CFU/ml medians were conducted using the Kruskal-Wallis test, followed by Bonferroni’s post-hoc test. Differences in pellet R-values between baseline and two weeks later were evaluated using a paired t-test. For comparisons of mean R-values and ΔR among groups, one-way ANOVA with Tukey’s post-hoc test was performed. Statistical significance was set at p<0.05.

Results

1. Evaluation of water quality contamination immediately after disinfection and after two weeks of use

At baseline (immediately after routine disinfection), the CFU/ml levels in 3W, US, and CF were significantly lower, meeting the normal threshold, with no differences observed among the groups (p>0.05). However, after two weeks of use, the CFU/ml levels for 3W, US, and CF exceeded the threshold of 100 CFU/ml, with the highest contamination observed in the US group (Table 1). At baseline, the colonies in the 3W, US, and CF groups were barely visible. After two weeks of use, the colonies became distinctly visible in all groups. In the 3W and US groups, white and yellow colonies of varying sizes were observed, whereas in the CF group, white and yellow colonies of similar sizes were noted (Fig. 1).

Fig. 1. Comparison of contamination at baseline and after two weeks via bacterial culturing on R2A agar plates. 3W: 3-way syringe, US: ultrasonic scalers, CF: cup fillers.

Evaluation of Water Quality of 3-Way Syringes (3W), Ultrasonic Scaler (US), and Cup Fillers (CF)

Section Baseline 2 weeks later
3W 0.90 (0.60∼1.50) 970.00 (930.00∼1110.00)ab
US 17.20 (15.50∼20.60) 1,830.00 (1,660.00∼2,000.00)a
CF 0.70 (0.40∼0.90) 720.00 (710.00∼810.00)b
p-value 0.055 0.027

Values are presented as median (range).

p-values were obtained using the Kruskal-Wallis test.

a,bDifferent letters indicate statistically significant differences according to Bonferroni’s post-hoc test.



2. Comparison of colony biofluorescence by sampling time

The observation of colonies cultured from each sample revealed green, orange, and red fluorescent colonies in the 3W, US, and CF samples, respectively. In QLF-D imaging, the white and blue light reflected from the medium was observed as a circular pattern in the central area of the medium. At baseline, no colonies were observed on QLF-D imaging. However, after two weeks, colonies of varying sizes exhibiting green and red fluorescence were observed in the 3W samples. In US samples, most colonies displayed red fluorescence. In the CF samples, the colonies were relatively small, making it difficult to distinguish their colors without close observation; however, red fluorescence was detected (Fig. 2).

Fig. 2. Biofluorescence observation of colonies for each sample using quanti-tative light-induced fluorescence-digital. 3W: 3-way syringe, US: ultrasonic scalers, CF: cup fillers.

3. Observation and analysis of pellet fluorescence

QLF-D imaging of the pellets revealed that baseline R-values ranged from 40.33 to 67.00, with the R-value for US being significantly higher than those for 3W and CF (p<0.001). After two weeks of dental unit use, the R-values for pellets from all groups increased significantly, with notable differences observed among groups (p<0.001). Considering the baseline differences among groups, the analysis of ΔR showed that the change was significantly lower in CF compared to 3W and US (p<0.001) (Table 2). QLF-D fluorescent images also demonstrated a clear increase in the redness of the pellets after two weeks of use compared to that immediately after disinfection (Fig. 3).

Fig. 3. Biofluorescence observations of centrifuged pellets using quantitative light-induced fluorescence-digital. 3W: 3-way syringe, US: ultrasonic scalers, CF: cup fillers.

Comparison of R-values and ΔR of Pellets between Groups

Baseline 2 weeks later p-valuea ΔR
3W 41.44±3.71c 82.67±5.05c <0.001 41.22±6.12c
US 67.00±4.87d 114.78±18.10d <0.001 47.78±18.21c
CF 40.33±6.06c 56.33±8.32e <0.001 16.00±7.02d
p-valueb <0.001 <0.001 <0.001

Values are presented as or mean±standard deviation.

3W: 3-way syringe, US: ultrasonic scalers, CF: cup fillers.

ap-values were obtained from paired t-test between the R-values of baseline and 2 weeks later.

bp-values were obtained from one-way ANOVA.

c,d,eDifferent letters indicated statistically significant differences in Tukey’s post-hoc test.


Discussion

Due to their narrow-diameter tubing and extended water retention times, DUWLs are prone to microbial contamination, biofilm formation, and opportunistic pathogens. Furthermore, fluid flow, shear stress, antibiotic use, and oxygen concentration can influence microbial attachment15-18).

According to Korea’s dental infection control standard policy and procedure, the standard for bacterial counts in DUWLs is 100 CFU/ml. In contrast, the CDC recommends maintaining bacterial levels below 500 CFU/ml for non-surgical dental procedures DUWL5,14). In this study, we assessed water contamination levels based on domestic standards. The results showed no statistically significant differences in the level of water contamination in DUWLs immediately after disinfection, regardless of the sampling part, and the levels met domestic standards. Similarly, a previous review indicated that using approved disinfectants effectively reduces microbial levels in DUWLs to acceptable levels19). However, after two weeks, the water contamination levels in all samples exceeded both the domestic standards and the CDC’s recommended levels. The increase in contamination was visibly reflected in the intensified redness observed in the fluorescence images of the pellet using QLF-D. This result confirmed a clear relationship between the increased contamination levels within the waterlines over time and the increased redness intensity of the pellets. Dang et al.20) reported that the types of microorganisms identified differed depending on the specific dental department and treatments conducted, emphasizing the need to use disinfectants suitable for each department. The American Dental Association also recommended specific disinfectants for different dental clinics21). Considering that the disinfection effect of H2O2 did not last for two weeks in this study, it is suggested that it is necessary to adjust the disinfection period or switch to a disinfectant appropriate for the type of treatment.

Among the sampling sites, the highest microbial contamination and vital redness values were observed in the US when the dental unit was used for two weeks after disinfection. A previous study confirmed contamination levels of 8,420 CFU/ml in the US within a university laboratory used more than three times per month22). Additionally, research reported bacterial contamination levels reaching up to 2.0×105 CFU/ml within five days of installing a new dental unit23). These findings suggest that controlling microbial contamination among DUWLs in the US is particularly challenging, even when waterline disinfection is conducted uniformly in dental hygiene departments and laboratories. Biofilms formed on the surfaces of medical devices are not entirely removed even after disinfection, allowing the regrowth of microorganisms. Previous studies have also demonstrated that biofilms exhibit strong resistance to external disinfection and, as observed in this study, tend to promote microbial regrowth over time24). This is because the extracellular polymeric substances secreted by microorganisms form a biofilm that acts as a barrier, impeding the penetration of disinfectants24).

This study demonstrates that regular disinfection effectively reduces initial contamination in DUWLs but is insufficient for completely removing biofilms. Given the recommended domestic standard of 100 CFU/ml, the two-week disinfection cycle used in this experiment’s dental hygiene laboratory subjects requires improvement. Furthermore, to comply with DUWL standards, it is essential to use approved waterline disinfectants according to the manufacturer’s instructions and monitor water quality regularly. In the future, the QLF could be utilized in dental hospitals and clinics to monitor water contamination across various treatment environments. Further studies are required to evaluate the effectiveness of these strategies in these settings.

1. Limitation

This study served as a foundational investigation into whether QLF-D can be used to determine appropriate disinfection cycles and assess contamination levels in dental unit management. However, the study was limited by a small sample size and was conducted under controlled conditions that included specific temperature and humidity levels in a dental hygiene laboratory without adequately accounting for various environmental variables. Furthermore, non-chemical management methods for DUWLs have not been considered. Therefore, future research should be conducted under conditions that resemble actual dental and laboratory environments.

2. Conclusion

This study highlighted the importance and limitations of waterline disinfection in controlling microbial contamination in dental units. Regular disinfection with H2O2 effectively reduced initial microbial contamination and met the infection control standards. However, the number of microorganisms increased rapidly over time. The US exhibited the highest levels of microbial contamination and biofilm-related fluorescence intensity within the DUWLs. These findings underscore the need for robust and frequent disinfection protocols to achieve sustained microbial control, particularly for high-risk devices. In addition, dental professionals must identify and rigorously implement effective water quality monitoring methods to maintain a safe dental environment. This includes exploring stricter infection control guidelines such as replacing disinfectants when necessary, increasing the frequency of DUWL disinfection, and consistently adhering to these standards.

Acknowledgements

None.

Notes

Conflict of interest

Ji-Hyun Min has been journal manager of the Journal of Dental Hygiene Science since January 2023. Ji-Hyun Min was not involved in the review process of this editorial. Otherwise, no potential conflict of interest relevant to this article was reported. Seong-Chan Park, Sun-Young Han declare that she has no conflicts of interest.

Ethical approval

This article does not involve human subjects; it does not require IRB approval.

Author contributions

Conceptualization: Sun-Young Han and Seong-Chan Park. Data acquisition: Seong-Chan Park. Formal analysis: Ji-Hyun Min and Seong-Chan Park. Supervision: Sun-Young Han. Writing-original draft: Seong-Chan Park. Writing-review & editing: Ji-Hyun Min and Sun-Young Han.

Funding

None.

Data availability

Raw data is provided at the request of the corresponding author for reasonable reasons.

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