scholarly journals Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1869-1875 ◽  
Author(s):  
Shoji Takenaka ◽  
Harsh M. Trivedi ◽  
Audrey Corbin ◽  
Betsey Pitts ◽  
Philip S. Stewart
Keyword(s):  
Spatial Information ◽  
Capillary Flow ◽  
Temporal Patterns ◽  
Laser Scanning Microscopy ◽  
Antimicrobial Action ◽  
Confocal Laser ◽  
Green Fluorescence ◽  
Spatial And Temporal Patterns ◽  
Bulk Fluid ◽  
Oral Biofilms

ABSTRACT A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 μm/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.

scholarly journals Effect of Etching Procedures on the Adhesion of Biofilm-Coated Dentin

Materials ◽  
2020 ◽  
Vol 13 (12) ◽  
pp. 2762
Author(s):  
Bo-Kyung Jeon ◽  
Chang-Ha Lee ◽  
A Reum Kim ◽  
Seung Hyun Han ◽  
Hyun-Jung Kim ◽  
...  
Keyword(s):  
Bond Strength ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Failure Modes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Acid Etching ◽  
Dentin Surface ◽  
Biofilm Removal ◽  
Oral Biofilms

Oral biofilms coat all surfaces in the oral cavity including the exposed dentin surface. This study aimed to investigate biofilm removal by acid etching procedures and the effects of the residual biofilm on dentin surfaces on composite–dentin adhesion. Dentin discs were assigned to five groups: no biofilm formation (C); biofilm formation and no surface treatment (BF); biofilm formation and acid etching (BF-E); biofilm formation and acid etching followed by chlorhexidine soaking (BF-EC); and biofilm formation and rubbing with pumice, followed by acid etching (BF-RE). Biofilms were formed on saliva-precoated dentin discs by soaking the discs in Streptococcus mutans (S. mutans) suspension. Biofilm removal from the dentin surface was evaluated quantitatively and qualitatively by confocal laser scanning microscopy and scanning electron microscopy, respectively. To compare the bond strength of the biofilm-coated dentin discs with the surface treatments, specimens were assigned to four groups: no biofilm formation and acid etching (C-E); BF-E; BF-EC; and BF-RE. Assessments of the micro-shear bond strength and subsequent failure modes were performed. BF-E and BF-EC did not remove the biofilm, whereas BF-RE partially removed the biofilm attached to the dentin (p < 0.05). The bond strength of BF-RE was significantly higher than those of BF-E and BF-EC, but lower than that of C-E (p < 0.05). In conclusion, mechanical biofilm removal is recommended before etching procedures to enhance adhesion to the biofilm-coated dentin.

scholarly journals Spatial and Temporal Patterns of Biocide Action against Staphylococcus epidermidis Biofilms

2010 ◽  
Vol 54 (7) ◽  
pp. 2920-2927 ◽  
Author(s):  
William M. Davison ◽  
Betsey Pitts ◽  
Philip S. Stewart
Keyword(s):  
Quaternary Ammonium ◽  
Staphylococcus Epidermidis ◽  
Membrane Integrity ◽  
Temporal Patterns ◽  
Quaternary Ammonium Compound ◽  
Antimicrobial Action ◽  
Ammonium Compound ◽  
Spatial And Temporal Patterns ◽  
Cell Clusters ◽  
Biofilm Removal

ABSTRACT The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter−1 chlorine, 21 min for 50 mg liter−1 chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.

scholarly journals Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization

2009 ◽  
Vol 58 (10) ◽  
pp. 1359-1366 ◽  
Author(s):  
Ali Al-Ahmad ◽  
Marie Follo ◽  
Ann-Carina Selzer ◽  
Elmar Hellwig ◽  
Matthias Hannig ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Bacterial Colonization ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Actinomyces Naeslundii ◽  
Oral Biofilms

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7±13.8 %; 6 h: 20.0±16.6 %; 12 h: 24.7±16.1 %). However, ≤1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

scholarly journals Spatio-temporal development of cuticular ridges on leaf surfaces of Hevea brasiliensis alters insect attachment

2020 ◽  
Vol 7 (11) ◽  
pp. 201319
Author(s):  
Venkata A. Surapaneni ◽  
Georg Bold ◽  
Thomas Speck ◽  
Marc Thielen
Keyword(s):  
Hevea Brasiliensis ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Indirect Defence ◽  
Spatial And Temporal Patterns ◽  
Traction Forces ◽  
Strong Negative Correlation ◽  
Leaf Surfaces ◽  
Spatio Temporal

Cuticular ridges on plant surfaces can control insect adhesion and wetting behaviour and might also offer stability to underlying cells during growth. The growth of the plant cuticle and its underlying cells possibly results in changes in the morphology of cuticular ridges and may also affect their function. We present spatial and temporal patterns in cuticular ridge development on the leaf surfaces of the model plant, Hevea brasiliensis . We have identified, by confocal laser scanning microscopy of polymer leaf replicas, an acropetally directed progression of ridges during the ontogeny of Hevea brasiliensis leaf surfaces. The use of Colorado potato beetles (Leptinotarsa decemlineata) as a model insect species has shown that the changing dimensions of cuticular ridges on plant leaves during ontogeny have a significant impact on insect traction forces and act as an effective indirect defence mechanism. The traction forces of walking insects are significantly lower on mature leaf surfaces compared with young leaf surfaces. The measured walking traction forces exhibit a strong negative correlation with the dimensions of the cuticular ridges.

Impact of Hydrodynamics on Oral Biofilm Strength

2009 ◽  
Vol 88 (10) ◽  
pp. 922-926 ◽  
Author(s):  
E. Paramonova ◽  
O.J. Kalmykowa ◽  
H.C. van der Mei ◽  
H.J. Busscher ◽  
P.K. Sharma
Keyword(s):  
Laser Scanning ◽  
Periodontal Diseases ◽  
Single Species ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Oral Biofilm ◽  
Actinomyces Naeslundii ◽  
Mechanical Removal ◽  
Hydrodynamic Shear ◽  
Oral Biofilms

Mechanical removal of oral biofilms is ubiquitously accepted as the best way to prevent caries and periodontal diseases. Removal effectiveness strongly depends on biofilm strength. To investigate the influence of hydrodynamics on oral biofilm strength, we grew single- and multi-species biofilms of Streptococcus oralis J22, Actinomyces naeslundii TV14-J1, and full dental plaque at shear rates ranging from 0.1 to 50 1/sec and measured their compressive strength. Subsequently, biofilm architecture was evaluated by confocal laser scanning microscopy. Multi-species biofilms were stronger than single-species biofilms, with strength values ranging from 6 to 51 Pa and from 5 to 17 Pa, respectively. In response to increased hydrodynamic shear, biofilm strength decreased, and architecture changed from uniform carpet-like to more “fluffy” with higher thickness. S. oralis biofilms grown under variable shear of 7 and 50 1/sec possessed properties intermediate of those measured at the respective single shears.

scholarly journals Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

2011 ◽  
Vol 77 (10) ◽  
pp. 3413-3421 ◽  
Author(s):  
Rebecca Peyyala ◽  
Sreenatha S. Kirakodu ◽  
Jeffrey L. Ebersole ◽  
Karen F. Novak
Keyword(s):  
Laser Scanning ◽  
Bacterial Species ◽  
Disease Process ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Content Type ◽  
Oral Biofilms ◽  
Multispecies Biofilms

ABSTRACTOral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novelin vitromodel system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprisingStreptococcus gordonii,Streptococcus oralis, andStreptococcus sanguinis;S. gordonii,Actinomyces naeslundii, andFusobacterium nucleatum; orS. gordonii,F. nucleatum, andPorphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.

scholarly journals Novel Broad-Spectrum Antimicrobial Photoinactivation ofIn SituOral Biofilms by Visible Light plus Water-Filtered Infrared A

2014 ◽  
Vol 80 (23) ◽  
pp. 7324-7336 ◽  
Author(s):  
L. Karygianni ◽  
S. Ruf ◽  
M. Follo ◽  
E. Hellwig ◽  
M. Bucher ◽  
...  
Keyword(s):  
Visible Light ◽  
Laser Scanning ◽  
Treatment Approach ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Pharmaceutical Treatment ◽  
Oral Biofilms ◽  
Biofilm Bacteria ◽  
Bovine Enamel

ABSTRACTAntimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtainedin situ; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 μg ml−1toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm−2for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log10CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.

scholarly journals A Selective Ratiometric Fluorescent Probe for No-Wash Detection of PVC Microplastic

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1588
Author(s):  
Valeria Caponetti ◽  
Alexandra Mavridi-Printezi ◽  
Matteo Cingolani ◽  
Enrico Rampazzo ◽  
Damiano Genovese ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Direct Detection ◽  
Low Cost ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Intensity Ratio ◽  
Green Fluorescence ◽  
One Pot ◽  
Emerging Pollutant

Microplastics (MP) are micrometric plastic particles present in drinking water, food and the environment that constitute an emerging pollutant and pose a menace to human health. Novel methods for the fast detection of these new contaminants are needed. Fluorescence-based detection exploits the use of specific probes to label the MP particles. This method can be environmentally friendly, low-cost, easily scalable but also very sensitive and specific. Here, we present the synthesis and application of a new probe based on perylene-diimide (PDI), which can be prepared in a few minutes by a one-pot reaction using a conventional microwave oven and can be used for the direct detection of MP in water without any further treatment of the sample. The green fluorescence is strongly quenched in water at neutral pH because of the formation dimers. The ability of the probe to label MP was tested for polyvinyl chloride (PVC), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), poly methyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE). The probe showed considerable selectivity to PVC MP, which presented an intense red emission after staining. Interestingly, the fluorescence of the MP after labeling could be detected, under excitation with a blue diode, with a conventional CMOS color camera. Good selectivity was achieved analyzing the red to green fluorescence intensity ratio. UV–Vis absorption, steady-state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, fluorescence wide-field and confocal laser scanning microscopy allowed elucidating the mechanism of the staining in detail.

scholarly journals Spatiotemporal development of cuticular ridges on leaf surfaces of Hevea brasiliensis alters insect attachment

2020 ◽  
Author(s):  
Venkata A. Surapaneni ◽  
Georg Bold ◽  
Thomas Speck ◽  
Marc Thielen
Keyword(s):  
Hevea Brasiliensis ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Indirect Defence ◽  
Spatial And Temporal Patterns ◽  
Traction Forces ◽  
Strong Negative Correlation ◽  
Leaf Surfaces ◽  
Insect Attachment

AbstractCuticular ridges on plant surfaces can control insect adhesion and wetting behaviour and might also offer stability to underlying cells during growth. The growth of the plant cuticle and its underlying cells possibly results in changes in the morphology of cuticular ridges and may also affect their function. We present spatial and temporal patterns in cuticular ridge development on the leaf surfaces of the model plant, Hevea brasiliensis. We have identified, by confocal laser scanning microscopy of polymer leaf replicas, an acropetally directed progression of ridges during the ontogeny of Hevea brasiliensis leaf surfaces. The use of Colorado potato beetles (Leptinotarsa decemlineata) as a model insect species has shown that the changing dimensions of cuticular ridges on plant leaves during ontogeny have a significant impact on insect traction forces and act as an effective indirect defence mechanism. The traction forces of walking insects are significantly lower on mature leaf surfaces compared with young leaf surfaces. The measured walking traction forces exhibit a strong negative correlation with the dimensions of the cuticular ridges.

scholarly journals Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

2012 ◽  
Vol 78 (24) ◽  
pp. 8703-8711 ◽  
Author(s):  
L. Karygianni ◽  
M. Follo ◽  
E. Hellwig ◽  
D. Burghardt ◽  
M. Wolkewitz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Large Scale ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Bacterial Composition ◽  
Entire Area ◽  
Content Type ◽  
Oral Biofilms

ABSTRACTA microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grownin situon BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria,Streptococcusspp., andFusobacterium nucleatumwere stained using specific fluorescencein situhybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.

Sign in / Sign up

Export Citation Format

Share Document