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Image analysis steps used in ImageJ to calculate bacterial viability from a confocal image of biofilm with LIVE/DEAD stain. Images taken from a representative S. sanguinis biofilm cultured for 48 h (20 µm scale bar). See Supplementary Information to implement the automated analysis.
Source publication
Quantifying biofilm formation on surfaces is challenging because traditional microbiological methods, such as total colony-forming units (CFUs), often rely on manual counting. These are laborious, resource intensive techniques, more susceptible to human error. Confocal laser scanning microscopy (CLSM) is a high-resolution technique that allows 3D v...
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... assess the reliability and accuracy of the automated protocol developed (Fig. 1), a series of analyses were performed. This included sensitivity and specificity analysis 28 , a comparison with traditional microbiological techniques and the application of the protocol to a variety of bacterial species with varying morphologies (Fig. ...
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... and materials scientists. Therefore, the analysis protocol was written in the open-source software ImageJ and the method requires no preliminary image preparation or modification. The ImageJ macro, alongside a fully detailed description of how to execute the algorithm to enable researchers to implement this protocol, is available in Fig. 1 and the Supplementary Information. The algorithm was effective and accurate on a range of biofilms, including different bacterial morphologies, such as cocci (S. sanguinis) and rod-shaped (L. casei) bacteria, and different biofilm ages (from 24 h up to 7 days). A full workflow is provided alongside a series of validation methods in ...
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... studies that use open-source software have not included the code to allow for easy replication by other scientists wishing to use their method. One of the key strengths of this work is that a copy of the code, instructions on how to implement it and an overview of the image analysis protocol are all provided to ensure reproducibility (see Fig. 1 and Supplementary Information) 14 . This allows for users to understand the code and easily modify it to fit the data being analysed. For example, if a different staining protocol is used, the pre-processing steps can be removed or adjusted so as not to account for the issues identified with the FilmTracer™ LIVE/DEAD® Biofilm Viability ...
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... with the dead (red) bacteria in the image, and finally subtracting to find the number of pixels corresponding with live bacteria. The live bacteria were quantified as a percentage of the total bacteria in each image. The image analysis method was carried out using Fiji (ImageJ, US National Institutes of Health, Bethesda, Maryland, USA) ( Fig. 1). This was chosen due to it being open-source software, and therefore freely available. It should be noted that this macro calculates viability based on the assumption that the image contains a single-species biofilm, and therefore the area of red and green bacteria are proportional to the number of red and green bacteria, respectively. ...
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Citations
... Images were analyzed using COMSTAT, an image processing application built on top of Matlab 6.5's built-in image processing tools, to determine biovolume (m 3 /m 2 ) in the biofilm layer. A total of 10 locations on each anode were chosen for microscopic analysis [20]. Biovolumes of cell biomass were calculated and plotted for assessment separately for each anode type. ...
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... Most studies that evaluate the efficacy of novel potential antibiofilm agents use standard techniques (crystal violet assay, CFU plate counting, tetrazolium salts, or resazurinbased methods) accompanied by a visualization method for the confirmation and complementation of results. Direct imaging of biofilms allows the assessment of structural changes after treatment, while fluorescence staining permits selective labeling, providing additional information regarding biofilm composition and the viability/vitality of the microorganisms [10]. However, the fast and accurate visualization of these methods provide them with the potential to be used on their own as alternatives of conventional methods. ...
Viability and vitality assays play a crucial role in assessing the effectiveness of novel therapeutic approaches, with stain-based methods providing speed and objectivity. However, their application in yeast research lacks consensus. This study aimed to assess the performance of four common dyes on C. parapsilosis planktonic cells as well as sessile cells that form well-structured biofilms (treated and not treated with amphotericin B). Viability assessment employed Syto-9 (S9), thiazole orange (TO), and propidium iodide (PI). Metabolic activity was determined using fluorescein diacetate (FDA) and FUN-1. Calcofluor white (CW) served as the cell visualization control. Viability/vitality percentage of treated samples were calculated for each dye from confocal images and compared to crystal violet and PrestoBlue results. Heterogeneity in fluorescence intensity and permeability issues were observed with S9, TO, and FDA in planktonic cells and biofilms. This variability, influenced by cell morphology, resulted in dye-dependent viability/vitality percentages. Notably, PI and FUN-1 exhibited robust C. parapsilosis staining, with FUN-1 vitality results comparable to PrestoBlue. Our finding emphasizes the importance of evaluating dye permeability in yeast species beforehand, incorporating cell visualization controls. An improper dye selection may lead to misinterpreting treatment efficacy.
... The application of big data and machine learning in biofilm engineering can also streamline the optimization of bioprocesses [50]. Traditional trial-and-error approaches to improve biofilm-related technologies can be time-consuming and resource-intensive [51]. Machine learning models, on the other hand, can efficiently analyze vast datasets and identify optimal conditions for enhanced biofilm performance [52]. ...
Biofilms exert a profound impact on various facets of human life. Positive instances of biofilm usage involve their capacity to immobilize pollutants such as heavy metals, while adverse cases result in infections like urinary tract infections. Therefore, the study of biofilm engineering emerges as crucial. Employing a bibliographic research approach, this paper delves into biofilm engineering, identifying key species like Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, among others. The investigation also unveils major research subjects and corresponding institutions dedicated to biofilm research. A comprehensive understanding of biofilm engineering holds profound implications for advancing knowledge in this domain.
... The EEM analysis was performed from 200 to 450 nm for excitation spectra and 250 to 550 nm for emission spectra at 5-nm intervals (Wei et al., 2022) (F-4600, Hitachi, Japan). The biofilms on the fouled membranes were stained with SYTO9 (ex 480/em 500)/PI (ex 490/em 635) to detect the ratio of live/dead cells and biofilm thickness Mountcastle et al., 2021). The microbial biomass was collected from the remaining fouled membrane and frozen at − 80 • C before being delivered to Majorbio Biopharm Technology Co., Ltd. ...
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anthranilate (MA) was prepared. It exhibited enhanced anti-biofouling performance than the exogenous addition
of QSIs, showing long-term stability and alleviating 22 % decrease in membrane flux compared with the virgin membrane. The results observed that dominant bacteria Epsilon- and Gamma-proteobacteria (Shewanella, Olleya, Colwellia, and Arcobacter), which are significantly related to (P≤0.01) the metabolic products (i.e., polysaccharides, proteins and eDNA), are reduced by over 80 % on the MA m membrane. Additionally, the introduction of MA has a more significant impact on the QS signal-sensing pathway through binding to the active site of the transmembrane sensor receptor. It effectively reduces the abundance of genes encoding QS and extra-cellular polymeric substance (EPS) (exopolysaccharides (i.e., galE and nagB) and amino acids (i.e., ilvE, metH, phhA, and serB)) by up to 50 % and 30 %, respectively, resulting in a reduction of EPS by more than 50 %, thereby limiting the biofilm formation on the QSI-modified membrane. This study provides novel insights into the potential of QSIs to control consortial biofilm formation in practical SWRO applications.
... CLSM, coupled with live/dead staining, provides a fluorescence assay of bacterial viability. The surface properties of implanted materials also influence biofilm formation, and investigations utilizing CLSM complement microbiological approaches to elucidate biofilm matrix stability and function (Mountcastle et al., 2021;LewisOscar et al., 2021). ...
Infectious diseases, including urinary tract infections (UTIs), significantly impact global health, diminishing the quality of life and increasing clinical and economic burdens. Concerns arise due to the potential development of antimicrobial resistance (AMR) facilitated by biofilm formation. Nanotechnology, specifically silver and silver nanoparticles (1–100 nm), proves effective against bacterial strains, making them valuable in the biomedical field. Advances in microscopic imaging techniques, such as Confocal Laser Scanning Microscopy (CLSM), enhance our understanding of biofilms. This study aims to assess the effectiveness and optimal concentration of gold and silver nanoparticles in combating biofilms formed by uropathogenic Escherichia coli (UPEC) through CLSM analysis. The research comprises the following stages: rejuvenation of bacterial isolates and inoculum preparation, testing the anti-biofilm activity of gold and silver nanoparticles at concentrations of 25, 50, 75, and 100 ppm against UPEC using CLSM analysis. The findings reveal that both gold and silver nanoparticles exhibit anti-biofilm activity against UPEC at concentrations of 25, 50, 75, and 100 ppm, as confirmed by CLSM analysis. The optimal concentration for anti-biofilm activity of gold and silver nanoparticles against UPEC is identified as 100 ppm.
... Propidium iodide-stained cells, and SYTO 9-stained cells were visualized using Texas Red and GFP filters, respectively. The resulting TIFF images were analyzed and quantified using the Biofilm Viability Checker plugin for Fiji (ImageJ ™ ) image analysis software [36], which determined the proportion of live and dead bacteria. ...
Background
While antibiotics remain our primary tools against microbial infection, increasing antibiotic resistance (inherent and acquired) is a major detriment to their efficacy. A practical approach to maintaining or reversing the efficacy of antibiotics is the use of other commonly used therapeutics, which show synergistic antibacterial action with antibiotics. Here, we investigated the extent of antibacterial synergy between the antibiotic gentamicin and the anti-inflammatory ketorolac regarding the dynamics of biofilm growth, the rate of acquired resistance, and the possible mechanism of synergy.
Methods
Control (ATCC 12600, ATCC 35984) and clinical strains (L1101, L1116) of Staphylococcus aureus and Staphylococcus epidermidis with varying antibiotic susceptibility profiles were used in this study to simulate implant-material associated low-risk and high-risk biofilms in vitro. The synergistic action of gentamicin sulfate (GS) and ketorolac tromethamine (KT), against planktonic staphylococcal strains were determined using the fractional inhibitory concentration measurement assay. Nascent (6 h) and established (24 h) biofilms were grown on 316L stainless steel plates and the synergistic biofilm eradication activity was determined and characterized using adherent bacteria count, minimum biofilm eradication concentration (MBEC) measurement for GS, visualization by live/dead imaging, scanning electron microscopy, gene expression of biofilm-associated genes, and bacterial membrane fluidity assessment.
Results
Gentamicin-ketorolac (GS-KT) combination demonstrated synergistic antibacterial action against planktonic Staphylococci. Control and clinical strains showed distinct biofilm growth dynamics and an increase in biofilm maturity was shown to confer further resistance to gentamicin for both ‘low-risk’ and ‘high-risk’ biofilms. The addition of ketorolac enhanced the antibiofilm activity of gentamicin against acquired resistance in staphylococcal biofilms. Mechanistic studies revealed that the synergistic action of gentamicin–ketorolac interferes with biofilm morphology and subverts bacterial stress response altering bacterial physiology, membrane dynamics, and biofilm properties.
Conclusion
The results of this study have a significant impact on the local administration of antibiotics and other therapeutic agents commonly used in the prevention and treatment of orthopaedic infections. Further, these results warrant the study of synergy for the concurrent or sequential administration of non-antibiotic drugs for antimicrobial effect.
... CLSM, coupled with live/dead staining, provides a fluorescence assay of bacterial viability. The surface properties of implanted materials also influence biofilm formation, and investigations utilizing CLSM complement microbiological approaches to elucidate biofilm matrix stability and function (Mountcastle et al., 2021;LewisOscar et al., 2021). ...
Infectious diseases, including urinary tract infections (UTIs), significantly impact global health, diminishing the quality of life and increasing clinical and economic burdens. Concerns arise due to the potential development of antimicrobial resistance (AMR) facilitated by biofilm formation. Nanotechnology, specifically silver and silver nanoparticles (1–100 nm), proves effective against bacterial strains, making them valuable in the biomedical field. Advances in microscopic imaging techniques, such as Confocal Laser Scanning Microscopy (CLSM), enhance our understanding of biofilms. This study aims to assess the effectiveness and optimal concentration of gold and silver nanoparticles in combating biofilms formed by uropathogenic Escherichia coli (UPEC) through CLSM analysis. The research comprises the following stages: rejuvenation of bacterial isolates and inoculum preparation, testing the anti-biofilm activity of gold and silver nanoparticles at concentrations of 25, 50, 75, and 100 ppm against UPEC using CLSM analysis. The findings reveal that both gold and silver nanoparticles exhibit anti-biofilm activity against UPEC at concentrations of 25, 50, 75, and 100 ppm, as confirmed by CLSM analysis. The optimal concentration for anti-biofilm activity of gold and silver nanoparticles against UPEC is identified as 100 ppm.
... Given the increasing concerns, there is a pressing need for detection methods that are rapid, highly sensitive, and straightforward [3,[7][8][9]. Over time, researchers have developed a variety of effective detection methods, such as colony counting [10][11][12], polymerase chain reaction (PCR) [13], and enzyme-linked immunosorbent assay (ELISA) [14]. While these traditional methods exhibit commendable sensitivity, their prolonged experimental duration coupled with intricate and cumbersome protocols substantially limit their practical adoption [15]. ...
A direct and ultra-sensitive surface-enhanced Raman scattering (SERS) immunoassay method is introduced for the detection of Escherichia coli and Staphylococcus aureus. This methodology is based on a sandwich-structured complex probe (SCP) mechanism, combined with target-induced strand displacement. Moreover, by leveraging the amplified SERS signal from gold nanoparticles (AuNPs) corresponding to an increase in bacterial count, we achieve quantitative determination. The SCP demonstrates remarkable specificity, sensitivity, and anti-interference capability in bacterial detection. The detection limits for both bacterial strains are as low as 10 CFU/mL. In our selectivity tests, all peak intensities had standard deviations (n = 3) below 6%. Recoveries in normal human serum were 101–110% for E. coli and 96–101% for S. aureus. In milk, the recoveries were 102–105% for E. coli and 100–105% for S. aureus, respectively, demonstrating a high level of accuracy and resistance to interference. In addition, the SCP offers a dual-detection capability, enabling simultaneous diagnosis of multiple targets, which greatly simplifies the testing procedure. The findings underscore that this immunoassay platform fulfills the demand for rapid and precise pathogenic bacterial diagnosis, holding substantial potential for practical applications.
Graphical abstract
... 10. All images are processed using ImageJ [30]. ImageJ was used to combine/overlay two images [e.g., DAPI and Thioflavin T (green)] into a single image to generate a representative image as shown in Figure 3. ...
Campylobacter jejuni, a zoonotic foodborne pathogen, is the worldwide leading cause of acute human bacterial gastroenteritis. Biofilms are a significant reservoir for survival and transmission of this pathogen, contributing to its overall antimicrobial resistance. Natural compounds such as essential oils, phytochemicals, polyphenolic extracts, and D-amino acids have been shown to have the potential to control biofilms formed by bacteria, including Campylobacter spp. This work presents a proposed guideline for assessing and characterizing bacterial biofilm formation in the presence of naturally occurring inhibitory molecules using C. jejuni as a model. The following protocols describe: i) biofilm formation inhibition assay, designed to assess the ability of naturally occurring molecules to inhibit the formation of biofilms; ii) biofilm dispersal assay, to assess the ability of naturally occurring inhibitory molecules to eradicate established biofilms; iii) confocal laser scanning microscopy (CLSM), to evaluate bacterial viability in biofilms after treatment with naturally occurring inhibitory molecules and to study the structured appearance (or architecture) of biofilm before and after treatment.
... To see dense biofilm samples in detail, image-processing methods are utilized for the quantitative investigation of biofilms. This method was also used to assess bacterial viability and biofilm availability on transparent and opaque surfaces [31]. Fluorescent dyes are applied to biofilms to distinguish between live and dead bacteria, allowing bacteria to be differentiated according to the permeability of the cytoplasmic membrane. ...
Caries is a common dental problem brought on by factors like excessive sugar consumption, poor oral hygiene, and the presence of microorganisms in the mouth. This dental pathology is treated with a variety of filling materials, including tooth-colored direct resin dental composite (RDC), glass ionomer cement (GIC), and dental amalgam (also known as silver filling). RDC is the most preferred filling material in dental clinics due to its excellent esthetics and minimal tooth preparation, making it the need of the modern era. However, antimicrobial agents were added to this material in order to enhance its ability to prevent secondary caries. The antibacterial activity of RDC has been tested using a variety of methods, but testing protocols have been found to vary. Thusly, the point of this article is to examine the disparity in the strategy involved by specialists for testing the antibacterial properties of RDCs.
