Interactions of microorganisms within a urinary catheter polymicrobial biofilm model.

Biofilms are often polymicrobial in nature, which can impact their behavior and overall structure, often resulting in an increase in biomass and enhanced antimicrobial resistance. Using plate counts and locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH), we studied the interactions of four species commonly associated with catheter-associated urinary tract infections (CAUTI): Enterococcus faecalis, Escherichia coli, Candida albicans, and Proteus mirabilis. Eleven combinations of biofilms were grown on silicone coupons placed in 24-well plates for 24 h, 37°C, in artificial urine medium (AUM). Results showed that P. mirabilis was the dominant species and was able to inhibit both E. coli and C. albicans growth. In the absence of P. mirabilis, an antagonistic relationship between E. coli and C. albicans was observed, with the former being dominant. E. faecalis growth was not affected in any combination, showing a more mutualistic relationship with the other species. Imaging results correlated with the plate count data and provided visual verification of species undetected using the viable plate count. Moreover, the three bacterial species showed overall good repeatability SD (Sr ) values (0.1-0.54) in all combinations tested, whereas C. albicans had higher repeatability Sr values (0.36-1.18). The study showed the complexity of early-stage interactions in polymicrobial biofilms. These interactions could serve as a starting point when considering targets for preventing or treating CAUTI biofilms containing these species.

Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo-activated chlorine dioxide treatment.

AIM: To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. METHODS AND RESULTS: A photo-activated chlorine dioxide treatment (Photo ClO2 ) was tested in two independent experiments against thick (>100 µm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. CONCLUSIONS: Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z-slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. SIGNIFICANCE AND IMPACT OF STUDY: There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.

Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications.

Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.

Simulated aging of draught beer line tubing increases biofilm contamination.

Craft brewing is continually gaining popularity in the United States. Craft brewers are committed to producing a wide variety of products and have a vested interest in product quality. Therefore, these brewers have the expectation that the beer poured at the tap will match the quality product that left the brewery. The presence of biofilm in draught lines is hypothesized as a contributing factor when this expectation is not achieved. Clean in place strategies based on the Sinner's Circle of Cleaning are used to remediate organic and inorganic accumulation in beer draught lines, including controlling biofilm accumulation. A study was conducted to determine if repeated exposure to chemical cleaning of vinyl beer tubing impacted biofilm growth, kill/removal, and subsequent regrowth of a mixed species biofilm. The tubing was conditioned to simulate one, two, and five years of use. The data collected demonstrates a clear trend between simulated age of the tubing and biofilm accumulation on the surface. Bacterial log densities ranged from 5.6 Log10(CFU/cm2) for the new tubing to 6.6 Log10(CFU/cm2) for tubing aged to simulate five years of use. The counts for the yeast were similar. Caustic cleaning of the tubing, regardless of starting biofilm coverage, left less than 2.75 Log10(CFU/cm2) viable bacteria and yeast cells remaining on the tubing surface. This demonstrated the effectiveness of the caustic at controlling biofilm accumulation in the simulated beer draught line. The biofilm that accumulated in the five-year aged tubing was able to recover more quickly, reaching 3.6 Log10(CFU/cm2) within 24 h indicating the treatment did not fully eradicate the biofilm, suggesting that the strong chemistry used in this study would cease to be as effective over time.

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle.

There has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.

Coupon position does not affect Pseudomonas aeruginosa and Staphylococcus aureus biofilm densities in the CDC biofilm reactor.

The CDC Biofilm Reactor method is the standard biofilm growth protocol for the validation of US Environmental Protection Agency biofilm label claims. However, no studies have determined the effect of coupon orientation within the reactor on biofilm growth. If positional effects have a statistically significant impact on biofilm density, they should be accounted for in the experimental design. Here, we isolate and quantify biofilms from each possible coupon surface in the reactor to quantitatively determine the positional effects in the CDC Biofilm Reactor. The results showed no statistically significant differences in viable cell density across different orientations and vertical positions in the reactor. Pseudomonas aeruginosa log densities were statistically equivalent among all coupon heights and orientations. While the Staphylococcus aureus cell growth showed no statistically significant differences, the densities were not statistically equivalent among all coupon heights and orientations due to the variability in the data. Structural differences were observed between biofilms on the high-shear baffle side of the reactor compared to the lower shear glass side of the reactor. Further studies are required to determine whether biofilm susceptibility to antimicrobials differs based on structural differences attributed to orientation.

Guidelines for the statistical analysis of a collaborative study of a laboratory method for testing disinfectant product performance.

This paper presents statistical techniques suitable for analyzing a collaborative study (multilaboratory study or ring trial) of a laboratory disinfectant product performance test (DPPT) method. Emphasis is on the assessment of the repeatability, reproducibility, resemblance, and responsiveness of the DPPT method. The suggested statistical techniques are easily modified for application to a single laboratory study. The presentation includes descriptions of the plots and tables that should be constructed during initial examination of the data, including a discussion of outliers and QA checks. The statistical recommendations deal with evaluations of prevailing types of DPPTs, including both quantitative and semiquantitative tests. The presentation emphasizes tests in which the disinfectant treatment is applied to surface-associated microbes and the outcome is a viable cell count; however, the statistical guidelines are appropriate for suspension tests and other test systems. The recommendations also are suitable for disinfectant tests using any microbe (vegetative bacteria, virus, spores, etc.) or any disinfectant treatment. The descriptions of the statistical techniques include either examples of calculations based on published data or citations to published calculations. Computer code is provided in an appendix.

Systematic exploration of natural and synthetic flavonoids for the inhibition of Staphylococcus aureus biofilms.

When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspended bacteria and represent more feasible antibacterial candidates.

α,α-disubstituted ß-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal.

Bacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted ß-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Da. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 µM acted as fast as 100 µM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted ß-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms.

Mitigation and use of biofilms in space for the benefit of human space exploration.

Biofilms are self-organized communities of microorganisms that are encased in an extracellular polymeric matrix and often found attached to surfaces. Biofilms are widely present on Earth, often found in diverse and sometimes extreme environments. These microbial communities have been described as recalcitrant or protective when facing adversity and environmental exposures. On the International Space Station, biofilms were found in human-inhabited environments on a multitude of hardware surfaces. Moreover, studies have identified phenotypic and genetic changes in the microorganisms under microgravity conditions including changes in microbe surface colonization and pathogenicity traits. Lack of consistent research in microgravity-grown biofilms can lead to deficient understanding of altered microbial behavior in space. This could subsequently create problems in engineered systems or negatively impact human health on crewed spaceflights. It is especially relevant to long-term and remote space missions that will lack resupply and service. Conversely, biofilms are also known to benefit plant growth and are essential for human health (i.e., gut microbiome). Eventually, biofilms may be used to supply metabolic pathways that produce organic and inorganic components useful to sustaining life on celestial bodies beyond Earth. This article will explore what is currently known about biofilms in space and will identify gaps in the aerospace industry's knowledge that should be filled in order to mitigate or to leverage biofilms to the advantage of spaceflight.

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization.

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.

Evaluation and remediation of bulk soap dispensers for biofilm.

Recent studies evaluating bulk soap in public restroom soap dispensers have demonstrated up to 25% of open refillable bulk-soap dispensers were contaminated with ~ 6 log(10)(CFU ml(-1)) heterotrophic bacteria. In this study, plastic counter-mounted, plastic wall-mounted and stainless steel wall-mounted dispensers were analyzed for suspended and biofilm bacteria using total cell and viable plate counts. Independent of dispenser type or construction material, the bulk soap was contaminated with 4-7 log(10)(CFU ml(-1)) bacteria, while 4-6 log(10)(CFU cm(-2)) biofilm bacteria were isolated from the inside surfaces of the dispensers (n = 6). Dispenser remediation studies, including a 10 min soak with 5000 mg l(-1) sodium hypochlorite, were then conducted to determine the efficacy of cleaning and disinfectant procedures against established biofilms. The testing showed that contamination of the bulk soap returned to pre-test levels within 7-14 days. These results demonstrate biofilm is present in contaminated bulk-soap dispensers and remediation studies to clean and sanitize the dispensers are temporary.

Interlaboratory evaluations of a standardized quantitative test method for determining the bactericidal and tuberculocidal efficacy of antimicrobial substances on hard non-porous surfaces.

The development, validation, and use of new quantitative methodologies for testing the effectiveness of antimicrobial products are necessary to meet the regulatory challenges associated with an ever-changing marketplace, novel product claims, new infection control practices, and the emergence of new clinical pathogens. A series of four interlaboratory studies were conducted in a standardized manner on an interim quantitative method for testing liquid treatments against bacteria to assess its statistical performance. The Quantitative Method, a derivative of ASTM E2197, is designed to enumerate the number of viable microbes remaining on a test carrier following exposure to a liquid antimicrobial treatment; a log10 reduction (LR) in viable bacteria is calculated based on the difference between the mean log10 density values of the untreated control and treated carriers. The Quantitative Method uses 1 cm diameter disks (carriers) of brushed stainless steel as the material to represent a hard, non-porous surface. The LR value is used as the measure of product effectiveness, where higher LR values are indicative of greater microbial kill. The test microbes were Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium terrae. The liquid antimicrobial treatments used in these studies were highly relevant to those in the marketplace and provided a wide range of mean LR outcomes. The focus of the statistical assessment was on the repeatability of the LRs across experiments within a lab (Sr) and the reproducibility of the LRs across labs (SR). Due to the additional sources of variability, the SR is expected to be higher than the variability within a laboratory (Sr); this was observed in the studies reported here. Across the studies, the Sr values for LR were small (i.e., less than 0.84), most notably for treatments generating high mean LRs (5 or above) where the Sr was as small as 0.12. Overall, the SR values ranged from 0.227 to 1.217. Only three of the twenty-four treatment combinations over the study period resulted in SR values above 1.0 - the associated LRs for the three treatments ranged from 2.22 to 3.26. Antimicrobial treatments with a LR of 4.5 or higher exhibited SR of 0.561 or less. The statistical attributes reported here for the draft Quantitative Method when used to test P. aeruginosa, S. aureus, and M. terrae provide information for decision makers when considering the method as a candidate regulatory procedure. The data and statistical analyses contained in this report are historical in nature and provide useful baseline information for individuals conducting additional technical review of the method. Based on the data, the Quantitative Method displays a statistical profile consistent with other standard methods approved by standard-setting organizations where method performance data are available.

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

The biofilm life cycle: expanding the conceptual model of biofilm formation.

Bacterial biofilms are often defined as communities of surface-attached bacteria and are typically depicted with a classic mushroom-shaped structure characteristic of Pseudomonas aeruginosa. However, it has become evident that this is not how all biofilms develop, especially in vivo, in clinical and industrial settings, and in the environment, where biofilms often are observed as non-surface-attached aggregates. In this Review, we describe the origin of the current five-step biofilm development model and why it fails to capture many aspects of bacterial biofilm physiology. We aim to present a simplistic developmental model for biofilm formation that is flexible enough to include all the diverse scenarios and microenvironments where biofilms are formed. With this new expanded, inclusive model, we hereby introduce a common platform for developing an understanding of biofilms and anti-biofilm strategies that can be tailored to the microenvironment under investigation.

Characterizing the Shearing Stresses within the CDC Biofilm Reactor Using Computational Fluid Dynamics.

Shearing stresses are known to be a critical factor impacting the growth and physiology of biofilms, but the underlying fluid dynamics within biofilm reactors are rarely well characterized and not always considered when a researcher decides which biofilm reactor to use. The CDC biofilm reactor is referenced in validated Standard Test Methods and US EPA guidance documents. The driving fluid dynamics within the CDC biofilm reactor were investigated using computational fluid dynamics. An unsteady, three-dimensional model of the CDC reactor was simulated at a rotation rate of 125 RPM. The reactor showed turbulent structures, with shear stresses averaging near 0.365 ± 0.074 Pa across all 24 coupons. The pressure variation on the coupon surfaces was found to be larger, with a continuous 2-3 Pa amplitude, coinciding with the baffle passage. Computational fluid dynamics was shown to be a powerful tool for defining key fluid dynamic parameters at a high fidelity within the CDC biofilm reactor. The consistency of the shear stresses and pressures and the unsteadiness of the flow within the CDC reactor may help explain its reproducibility in laboratory studies. The computational model will enable researchers to make an informed decision whether the fluid dynamics present in the CDC biofilm reactor are appropriate for their research.

Biofilms vs. cities and humans vs. aliens - a tale of reproducibility in biofilms.

Biofilms are complex and dynamic structures that include many more components than just viable cells. Therefore, the apparently simple goal of growing reproducible biofilms is often elusive. One of the challenges in defining reproducibility for biofilm research is that different research fields use a spectrum of parameters to define reproducibility for their particular application. For instance, is the researcher interested in achieving a similar population density, height of biofilm structures, or function of the biofilm in a certain ecosystem/industrial context? Within this article we categorize reproducibility into four different levels: level 1, no reproducibility; level 2, standard reproducibility; level 3, potential standard reproducibility; and level 4, total reproducibility. To better understand the need for these different levels of reproducibility, we expand on the 'cities of microbes' analogy for biofilms by imagining that a new civilization has reached the Earth's outskirts and starts studying the Earth's cities. This will provide a better sense of scale and illustrate how small details can impact profoundly on the growth and behavior of a biofilm and our understanding of reproducibility.

Novel phenolic antimicrobials enhanced activity of iminodiacetate prodrugs against biofilm and planktonic bacteria.

Prodrugs are pharmacologically attenuated derivatives of drugs that undergo bioconversion into the active compound once reaching the targeted site, thereby maximizing their efficiency. This strategy has been implemented in pharmaceuticals to overcome obstacles related to absorption, distribution, and metabolism, as well as with intracellular dyes to ensure concentration within cells. In this study, we provide the first examples of a prodrug strategy that can be applied to simple phenolic antimicrobials to increase their potency against mature biofilms. The addition of (acetoxy)methyl iminodiacetate groups increases the otherwise modest potency of simple phenols. Biofilm-forming bacteria exhibit a heightened tolerance toward antimicrobial agents, thereby accentuating the need for new antibiotics as well as those, which incorporate novel delivery strategies to enhance activity toward biofilms.

Critical Capability Needs for Reduction of Transmission of SARS-CoV-2 Indoors.

Coordination of efforts to assess the challenges and pain points felt by industries from around the globe working to reduce COVID-19 transmission in the indoor environment as well as innovative solutions applied to meet these challenges is mandatory. Indoor infectious viral disease transmission (such as coronavirus, norovirus, influenza) is a complex problem that needs better integration of our current knowledge and intervention strategies. Critical to providing a reduction in transmission is to map the four core technical areas of environmental microbiology, transmission science, building science, and social science. To that end a three-stage science and innovation Summit was held to gather information on current standards, policies and procedures applied to reduce transmission in built spaces, as well as the technical challenges, science needs, and research priorities. The Summit elucidated steps than can be taken to reduce transmission of SARS-CoV-2 indoors and calls for significant investments in research to enhance our knowledge of viral pathogen persistence and transport in the built environment, risk assessment and mitigation strategy such as processes and procedures to reduce the risk of exposure and infection through building systems operations, biosurveillance capacity, communication form leadership, and stakeholder engagement for optimal response. These findings reflect the effective application of existing knowledge and standards, emerging science, and lessons-learned from current efforts to confront SARS-CoV-2.

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.