scispace - formally typeset
Search or ask a question
Reference EntryDOI

Growing and analyzing static biofilms

TL;DR: In this article, the early stages of biofilm formation are examined using static biofilm assays, which are suitable for either small or relatively large-scale studies and can be used individually or in combination for the study of biofilms.
Abstract: Many bacteria can exist as surface-attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small- or relatively large-scale studies. Unlike assays involving continuous-flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms.

Content maybe subject to copyright    Report

Citations
More filters
Journal ArticleDOI
TL;DR: It is concluded that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence factor, as well as being shown to bind specifically to turkey tracheal cells via western blot analysis.

10 citations

Book ChapterDOI
TL;DR: This paper focuses on the use of 96-well optically clear, polystyrene flat-bottom plate to study biofilm formation by Leptospira spp.
Abstract: Biofilm formation in microtiter plates is certainly the most commonly used method to grow and study biofilm. This simple design is very popular due to its high-throughput screening capacities, low cost, and easy handling. In the protocol described here, we focus on the use of 96-well optically clear, polystyrene flat-bottom plate to study biofilm formation by Leptospira spp. and quantify the biofilm formation by crystal violet (CV) staining. We also describe an alternative method, based on phase contrast image analysis that we believe is more suitable for accurately quantifying biofilm growth by reducing handling of this fragile structure.

10 citations

Journal ArticleDOI
TL;DR: In this paper, two Nacyl-homoserine lactones (AHLs)-degrading enzymes, obtained from Actinoplanes utahensis (namely AuAAC and AuAHLA), were used to determine the effects of degradation of QS signals in the biofilm formation, among other virulence factors, of a Pseudomonas aeruginosa strain isolated from a WWTP, assessing molecular mechanisms through transcriptomics.

10 citations

Journal ArticleDOI
TL;DR: The consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a Pseudomonas fluorescens Pf0-1 model.
Abstract: Microtiter plate-based bacterial biofilm assay is frequently used to study bacterial biofilm development and growth While this assay is simple and relatively high-throughput, it frequently shows difficulty in establishing robust biofilm attachment in the wells We report that the consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a Pseudomonas fluorescens Pf0-1 model Scanning electron microscopy imaging suggests that the treated surfaces could provide points of attachment to facilitate the recruitment of bacteria in the initial phase of biofilm colony establishment The sandblast treated polypropylene, but not polystyrene, plates were found suitable in studying the impact of flavonoid quercetin on the biofilm formation in Bacillus subtilis FB17 Further investigation revealed that due to the hydrophobicity of the polystyrene surfaces, a greater amount of quercetin was adsorbed on the plate surface, effectively lowering the concentration of the flavonoid in solution

10 citations

Dissertation
14 Mar 2017
TL;DR: Two optical methods are examined which utilised different properties of fluorescence to investigate pH microgradients within biofilms designed to mimic cariogenic dental plaque to gain a greater understanding of how the microbial ecology is affected.
Abstract: The oral cavity is the most complex and accessible microbial ecosystem in the human body. It is the entrance to the respiratory and gastrointestinal tracts and, as such is exposed to unique environmental constraints. The human mouth is home to a myriad of microorganisms, many of which are exclusively found in this unique habitat. These microbial inhabitants can establish themselves and thrive in this environment by attaching to the various surfaces of the oral cavity. Following attachment, they form three-dimensional, complex and highly-integrated microbial communities. Despite their complexity and natural fluctuations in environmental parameters, in health, these communities remain relatively stable over time. This stability is termed microbial homeostasis. Disruption of the microbial homeostasis occurs as a result of regular or prolonged challenges in the form of an altered environment. These disruptions favour a shift in the microbial populations, suppressing the metabolism of the beneficial inhabitants and allowing unfavourable microorganisms to thrive in the lack of competition. This change in the oral microbiota facilitates the progression from oral health to disease. Despite continuing research and development in preventative measures dental caries, characterised by localised dissolution of the dental hard tissues, remain one of the most prevalent disorders affecting man today. This decay occurs as a result of strong organic acids produced by the microbiota within dental plaque following exposure to fermentable carbohydrates. Furthermore, prolonged and regular exposure to acids suppresses the growth of 'beneficial' bacteria allowing acidogenic, aciduric microorganisms, such as Streptococcus mutans and lactobacilli to thrive in the lack of competition. The presence of these acidogenic microorganisms causes an increase in acid production and an increase in the duration of exposure to those acids. Although rarely life-threatening, they create an enormous economic burden to healthcare providers worldwide and cause significant physical and social impact on those affected, including diet, communication and self-esteem. Greater understanding is required to appreciate the dynamic relationship that exists between the environment, the microbiota and the host. To gain a greater understanding of how the microbial ecology is affected, it is necessary to be able to determine how pH changes during and following fermentation and the effect these perturbations have upon the microbial community. At present, the most commonly employed methods include the use of microelectrodes, whether through insertion into laboratory grown biofilm or incorporated within in vivo prosthetics devices. These methods are not without their drawbacks. In the act of measurement, microelectrodes are inserted into the biofilm resulting in, at least partial, disruption of the biofilm and this may have a detrimental effect on the results. In vivo prosthetics provides measurement at the biofilm interfaces and in physiological conditions, however almost certainly require partially dentate individuals and are difficult to use. Novel methods are required to investigate pH within biofilms which provide a multidimensional determination, including temporal, and do not cause a detrimental effect upon the biofilm. Here, I examine two optical methods which utilised different properties of fluorescence to investigate pH microgradients within biofilms designed to mimic cariogenic dental plaque. The two methods are dual-fluorophore, ratiometric, pH-sensitive nanosensors imaged through confocal laser scanning microscopy and SNARF®-4F 5-(and-6)-carboxylic acid imaged through time-correlated single-photon counting and fluorescence-lifetime imaging microscopy. The nanosensors were designed, produced and characterised prior to calibration. The nanosensors were applied to biofilms with limited success, likely due to poor penetration. The optical properties of SNARF®-4F 5-(and-6)-carboxylic acid were characterised, including the two-photon molecular excitation wavelength for use here. The fluorophore was calibrated and applied to bacterial sediment and biofilms and the localised environmental pH assessed following exposure to a fermentable carbohydrate to decrease the pH. Many of the drawbacks experienced with currently employed methods have been addressed by these methods, however further research and development is required.

10 citations


Cites background or methods from "Growing and analyzing static biofil..."

  • ...Polycarbonate and nitrocellulose membranes provide a flat, thin, smooth and biologically inert substratum upon which to grow bacterial biofilms (Merritt et al. 2005)....

    [...]

  • ................................................................................. 96 Figure 2.2 Schematic diagram of the CDFF when set-up....

    [...]

  • ................................................................................................................... 82 Figure 1.41 Side aspect view and components of the CDFF (Pratten & Wilson 1999)....

    [...]

  • ...The method was adapted from Growing and Analysing Static Biofilms, Current Protocols in Microbiology (Merritt et al. 2005) ....

    [...]

  • ................................................... 100 Figure 2.3 CDFF tools including A) tamping tool, B) PTFE pan removal tool, and C) PTFE plug recession tool....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: The optical densities of stained bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections.
Abstract: The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through a mouse model for foreign body infections and a rat model for catheter-induced endocarditis. The adherence measurements of animal passed strains remained the same as those of the laboratory-maintained parent strain. Spectrophotometric classification of coagulase-negative staphylococci into nonadherent and adherent categories according to these measurements had a sensitivity, specificity, and accuracy of 90.6, 80.8, and 88.4%, respectively. We examined a previously described collection of 127 strains of coagulase-negative staphylococci isolated from an outbreak of intravascular catheter-associated sepsis; strains associated with sepsis were more adherent than blood culture contaminants and cutaneous strains (P less than 0.001). We also examined a collection of 84 strains isolated from pediatric patients with cerebrospinal fluid (CSF) shunts; once again, pathogenic strains were more adherent than were CSF contaminants (P less than 0.01). Finally, we measured the adherence of seven endocarditis strains. As opposed to strains associated with intravascular catheters and CSF shunts, endocarditis strains were less adherent than were saprophytic strains of coagulase-negative staphylococci. The optical densities of bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections. Images

1,980 citations


"Growing and analyzing static biofil..." refers methods in this paper

  • ...While popularized in the mid-to-late 1990s (Mack et al., 1994; O’Toole et al., 1999), the assay in its typically used form is derived from a protocol published by Christensen et al. (1985)....

    [...]

Journal ArticleDOI
TL;DR: Results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.
Abstract: The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 μg of tobramycin ml−1or 1.0 μg of ciprofloxacin ml−1. After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 ± 0.18 for tobramycin and 1.42 ± 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 μm into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.

918 citations

Journal ArticleDOI
TL;DR: The results suggest that some other resistance mechanism is involved for both agents and contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin.
Abstract: The penetration of two antibiotics, ampicillin and ciprofloxacin, through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely suspended bacteria to killing by the antibiotics were also measured. Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium. The susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined. Antibiotic penetration through biofilms was measured by assaying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk. Parallel experiments were performed with a mutant K. pneumoniae strain in which beta-lactamase activity was eliminated. For wild-type K. pneumoniae grown in suspension culture, ampicillin and ciprofloxacin MICs were 500 and 0.18 microgram/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment at 10 times the MIC were 4.43 +/- 0.33 and 4.14 +/- 0.33 for ampicillin and ciprofloxacin, respectively. Biofilms of the same strain were much less susceptible, yielding log reductions in the number of CFU of -0.06 +/- 0.06 and 1.02 +/- 0.04 for ampicillin and ciprofloxacin, respectively, for the same treatment. The number of CFU in the biofilms after 24 h of antibiotic exposure was not statistically different from the number after 4 h of treatment. Ampicillin did not penetrate wild-type K. pneumoniae biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The concentration of ciprofloxacin reached the MIC throughout the biofilm within 20 min. Ampicillin penetrated biofilms formed by a beta-lactamase-deficient mutant. However, the biofilms formed by this mutant were resistant to ampicillin treatment, exhibiting a 0.18 +/- 0.07 log reduction in the number of CFU after 4 h of exposure and a 1.64 +/- 0.33 log reduction in the number of CFU after 24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the mutant strain to ampicillin and ciprofloxacin could not be accounted for by antibiotic inactivation or slow diffusion since these antibiotics fully penetrated the biofilms. These results suggest that some other resistance mechanism is involved for both agents.

885 citations


"Growing and analyzing static biofil..." refers methods in this paper

  • ...Colony biofilms Colony biofilms (see Basic Protocol 3) have typically been used for the purpose of determining antibiotic resistance (Anderl et al., 2000; Walters et al., 2003)....

    [...]

Book ChapterDOI
TL;DR: This article operationally defines a biofilm as bacteria that are attached to a surface in sufficient numbers to be detected macroscopically.
Abstract: Interest in the study of microbial biofilms has increased greatly in recent years due in large part to the profound impact biofilms have in clinical, industrial, and natural settings. Traditionally, the study of biofilms has been approached from an ecological or engineering perspective, using a combination of classical microbiology and advanced microscopy. We and others have begun to use genetic approaches to understand the development of these complex communities. To begin we must answer the question: What is a biofilm? This definition, by necessity, may be quite broad because it is clear that many organisms can attach to a variety of surfaces under diverse environmental conditions. Therefore, in the context of this article we will operationally define a biofilm as bacteria that are attached to a surface in sufficient numbers to be detected macroscopically.

820 citations

Journal ArticleDOI
TL;DR: The results demonstrate that the mutants were impaired in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.
Abstract: The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to production of Staphylococcus epidermidis biofilms, which are thought to contribute to virulence in biomaterial-related infections. We isolated Tn917 transposon mutants of biofilm-producing S. epidermidis 13-1, which were completely biofilm negative. In pulsed-field gel electrophoresis no obvious deletions of the mutants were noted. The Tn917 insertions of mutants M10 and M11 were located on different EcoRI fragments but on identical 60-kb SmaI and 17-kb BamHI chromosomal fragments. Linkage of transposon insertions of mutants M10 and M11 with the altered phenotype was demonstrated by phage transduction, whereas the several other mutants apparently represented spontaneous variants. In a primary attachment assay with polystyrene spheres, no significant difference between any of the mutants and the wild type could be detected. Cell clustering as an indication of intercellular adhesion, which is a prerequisite for accumulation in multilayered cell clusters, was not detected with any mutant. These results demonstrate that the mutants were impaired in the accumulative phase of biofilm production. Mutants M10 and M11 did not produce detectable amounts of a specific polysaccharide antigen (D. Mack, N. Siemssen, and R. Laufs, Infect. Immun. 60:2048-2057, 1992), whereas substantially reduced amounts of antigen were produced by the spontaneous variants. Hexosamine was determined as the major specific component of the antigen enriched by gel filtration of biofilm-producing S. epidermidis 1457 because almost no hexosamine was detected in material prepared from the isogenic biofilm-negative transductant 1457-M11, which differentiates the antigen from other S. epidermidis polysaccharide components. Our results provide direct genetic evidence for a function of the antigen in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.

343 citations


"Growing and analyzing static biofil..." refers methods in this paper

  • ...While popularized in the mid-to-late 1990s (Mack et al., 1994; O’Toole et al., 1999), the assay in its typically used form is derived from a protocol published by Christensen et al. (1985)....

    [...]