Assessment of three-dimensional biofilm models through direct comparison with confocal microscopy imaging

2004 ◽  
Vol 49 (11-12) ◽  
pp. 177-185 ◽  
Author(s):  
J.B. Xavier ◽  
C. Picioreanu ◽  
M.C.M. van Loosdrecht
Keyword(s):  
Structure Formation ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Experimental Conditions ◽  
Microscopy Imaging ◽  
Biofilm Growth ◽  
Biofilm Development ◽  
Biofilm Structure

The mathematical modeling of spatial biofilm formation that provides the capability to predict biofilm structure from first principles has been in development for the past six years. However, a direct and quantitative link between model predictions and the experimentally observed structure formation still remains to be established. This work assesses the capability of a state-of-the-art technique for three-dimensional (3D) modeling of biofilm structure, individual based modeling (IbM), to quantitatively describe the early development of a multispecies denitrifying biofilm. Model evaluation was carried out by comparison of predicted structure with that observed from two experimental datasets using confocal laser scanning microscopy (CLSM) monitoring of biofilm development in laboratory flowcells. Experimental conditions provided biofilm growth without substrate limitation, which was confirmed from substrate profiles computed by the model. 3D structures were compared quantitatively using a set of morphological parameters including the biovolume, filled-space profiles, substratum coverage, average thickness and normalized roughness. In spite of the different morphologies detectable in the two independent short-term experiments analyzed here, the model was capable of accurate fitting data from both experiments. Prediction of structure formation was precise, as expressed by the set of morphology parameters used.

scholarly journals High Rates of Conjugation in Bacterial Biofilms as Determined by Quantitative In Situ Analysis

1999 ◽  
Vol 65 (8) ◽  
pp. 3710-3713 ◽  
Author(s):  
Martina Hausner ◽  
Stefan Wuertz
Keyword(s):  
Laser Scanning ◽  
Nutrient Concentration ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Bacterial Biofilms ◽  
Conjugative Gene Transfer ◽  
Biofilm Structure

ABSTRACT Quantitative in situ determination of conjugative gene transfer in defined bacterial biofilms using automated confocal laser scanning microscopy followed by three-dimensional analysis of cellular biovolumes revealed conjugation rates 1,000-fold higher than those determined by classical plating techniques. Conjugation events were not affected by nutrient concentration alone but were influenced by time and biofilm structure.

Automated biofilm morphology quantification from confocal laser scanning microscopy imaging

2003 ◽  
Vol 47 (5) ◽  
pp. 31-37 ◽  
Author(s):  
J.B. Xavier ◽  
D.C. White ◽  
J.S. Almeida
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Biofilm Structure ◽  
User Friendly ◽  
Processing Steps

In spite of the immediate visual appeal of confocal laser scanning microscopy images, the extraction of accurate reconstitutions of biofilm morphology requires a lengthy and computational intensive succession of processing steps. However, once performed, it provides ample reward by enabling the quantitative study of biofilm structure. A software suite of image processing tools for full automation of biofilm morphology quantification was developed by integrating preprocessing, segmentation and morphology quantification operations. This software toolbox was implemented in a web server and a user friendly interface was developed to facilitate image submission, storage and sharing, its access being unrestricted for scientific applications. The image bioinformatics tool which results from the integration of the processing operations can be accessed at http://www.itqb.unl.pt:1111/clsmip/. Its use is described in this paper and is illustrated with an example of processing of experimental data describing the growth of a mixed species denitrifying biofilm.

Advances in mathematical modeling of biofilm structure

Biofilms ◽  
2004 ◽  
Vol 1 (4) ◽  
pp. 337-349 ◽  
Author(s):  
C. Picioreanu ◽  
J. B. Xavier ◽  
M. C. M. van Loosdrecht
Keyword(s):  
Mathematical Modeling ◽  
Laser Scanning ◽  
Hydrogen Transfer ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Anammox Bacteria ◽  
Microscopy Imaging ◽  
Dimensional Modeling ◽  
Biofilm Structure ◽  
Solute Species

Mathematical modeling of spatial biofilm structure has been in development for the past 10 years, its main goal being to derive the dynamics of biofilm structure from first-principle descriptions of the various physical, chemical and biological processes involved in biofilm formation. Early efforts described development of unrestricted monospecies consortia, often considering diffusion and reaction of a single solute species. Multi-dimensional modeling of biofilms has presently reached a stage where multi-species systems with any number of bacterial and solute species, reactions and arbitrary detachment scenarios may be readily implemented using a general-purpose software framework introduced recently. The present work presents motivations for the mathematical modeling of biofilm structure and provides an overview on major contributions to this field from pioneering efforts using cellular automata (CA) to more recent methods using the preferred individual-based modeling (IbM). Recent examples illustrate how biofilm models can be used to study the microbial ecology in: (a) development of multi-species nitrifying biofilms with anammox bacteria, (b) interspecies hydrogen transfer in anaerobic digestion methanogenic consortia, (c) competition between flock-formers and filamentous bacteria influenced by environmental conditions and its effect on morphology of activated sludge flocs, and (d) a two-species biofilm system with structured biomass describing extracellular polymeric substances (EPS) and internal storage compounds. As recent efforts from direct comparison of structure predicted by three-dimensional modeling with that observed by confocal laser scanning microscopy imaging of biofilms grown in laboratory flow cells show a good agreement of predicted structures, multi-dimensional modeling approaches presently constitute a mature and established methodology to enhance our understanding of biofilm systems.

scholarly journals Coaggregation by the Freshwater Bacterium Sphingomonas natatoria Alters Dual-Species Biofilm Formation

2009 ◽  
Vol 75 (12) ◽  
pp. 3987-3997 ◽  
Author(s):  
K. R. Min ◽  
A. H. Rickard
Keyword(s):  
Glass Surface ◽  
Laser Scanning ◽  
Micrococcus Luteus ◽  
Single Species ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Biofilm Growth ◽  
Biofilm Development ◽  
Glass Surfaces ◽  
Sphingomonas Natatoria

ABSTRACTCoaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacteriumSphingomonas natatoriato form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregatingS. natatoria2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partnerMicrococcus luteus2.13 cells,S. natatoria2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attachedM. luteus2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficientS. natatoria2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attachedM. luteus2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregatingS. natatoria2.1gfp cells or the coaggregation-deficient variantS. natatoria2.1COGgfp cells were coinoculated in flow cells with viableM. luteus2.13 cells and allowed to grow together for 96 h. CoaggregatingS. natatoria2.1gfp cells outcompetedM. luteus2.13 cells, and 96-h biofilms were composed predominantly ofS. natatoria2.1gfp cells. Conversely, when coaggregation-deficientS. natatoria2.1COGgfp cells were coinoculated withM. luteus2.13 cells, the 96-h biofilm contained few coaggregation-deficientS. natatoria2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregatingS. natatoria2.1 populations in dual-species biofilms through competitive interactions.

scholarly journals The role of surface adhesion on the macroscopic wrinkling of biofilms

2021 ◽  
Author(s):  
Steffen Geisel ◽  
Eleonora Secchi ◽  
Jan Vermant
Keyword(s):  
Laser Scanning ◽  
Three Dimensional ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microfluidic Channels ◽  
Channel Networks ◽  
Biofilm Growth ◽  
And Control

Biofilms, bacterial communities of cells encased by a self-produced matrix, exhibit a variety of three-dimensional structures. Specifically, channel networks formed within the bulk of the biofilm have been identified to play an important role in the colonies viability by promoting the transport of nutrients and chemicals. Here, we study channel formation and focus on the role of the adhesion of the biofilm matrix to the substrate in Pseudomonas aeruginosa biofilms grown under constant flow in microfluidic channels. We perform phase contrast and confocal laser scanning microscopy to examine the development of the biofilm structure as a function of the substrates surface energy. The formation of the wrinkles and folds is triggered by a mechanical buckling instability, controlled by biofilm growth rate and the film's adhesion to the substrate. The three-dimensional folding gives rise to hollow channels that rapidly increase the overall volume occupied by the biofilm and facilitate bacterial movement inside them. The experiments and analysis on mechanical instabilities for the relevant case of a bacterial biofilm grown during flow enable us to predict and control the biofilm morphology.

scholarly journals Fibrin Biopolymer Incorporated with Antimicrobial Agents: A Proposal for Coating Denture Bases

Materials ◽  
2021 ◽  
Vol 14 (7) ◽  
pp. 1618
Author(s):  
Helena Sandrini Venante ◽  
Ana Paula Chappuis-Chocano ◽  
Oscar Oswaldo Marcillo-Toala ◽  
Rafaela Alves da Silva ◽  
Rodrigo Moreira Bringel da Costa ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Alcoholic Extract ◽  
Base Surface ◽  
Biofilm Growth ◽  
Denture Stomatitis ◽  
Colony Forming Unit ◽  
Biofilm Development

The characteristics of the denture base surface, in combination with the oral environment, promote the colonization and development of Candida albicans biofilm, which is the main cause of denture stomatitis. This study evaluated the effectiveness of fibrin biopolymer with digluconate chlorhexidine or Punica granatum alcoholic extract to prevent C. albicans biofilm. Conventional heat polymerized and pre-polymerized poly(methyl methacrylate) (PMMA) circular specimens (10 × 2 mm) were fabricated (n = 504) and randomly divided into groups: no treatment (control—CT), fibrin biopolymer coating (FB), fibrin biopolymer with P. granatum (FBPg), or digluconate of chlorhexidine (FBCh) coating. The specimens were inoculated with C. albicans SC5314 (1 × 107 cells/mL) and incubated for 24, 48, and 72 h. Crystal violet and colony-forming unit assays were used to quantify the total biofilm biomass and biofilm-living cells. A qualitative analysis was performed using confocal laser scanning microscopy. Data obtained are expressed as means and standard deviations and were statistically analyzed using a three-way analysis of variance (α = 0.05). The FBPg and FBCh groups inhibited the growth of C. albicans biofilm in both PMMA materials analyzed, with FBCh performing better in all periods evaluated (p < 0.0001). The colony forming unit (CFU) assay showed that the FB group favored the C. albicans biofilm growth at 24 h and 48 h (p < 0.0001), with no differences with CT group at 72 h (p = 0.790). All groups showed an enhancement in biofilm development up to 72 h (p < 0.0001), except the FBCh group (p = 0.100). No statistical differences were found between the PMMA base materials (p > 0.050), except in the FB group (p < 0.0001). Fibrin biopolymer, albeit a scaffold for the growth of C. albicans, when combined with chlorhexidine digluconate or P. granatum, demonstrated excellent performance as a drug delivery system, preventing and controlling the formation of denture biofilm.

scholarly journals Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz H. D. Panariello ◽  
Justin K. Kindler ◽  
Kenneth J. Spolnik ◽  
Ygal Ehrlich ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Root Canal ◽  
Laser Scanning ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Electromagnetic Stimulation ◽  
Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

scholarly journals Importance of Initial Interfacial Steps during Chalcopyrite Bioleaching by a Thermoacidophilic Archaeon

2020 ◽  
Vol 8 (7) ◽  
pp. 1009
Author(s):  
Camila Safar ◽  
Camila Castro ◽  
Edgardo Donati
Keyword(s):  
High Temperatures ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Copper Recovery ◽  
Confocal Laser ◽  
Mineral Surface ◽  
Thermophilic Microorganisms ◽  
Valuable Metals ◽  
Refractory Mineral ◽  
Biofilm Development

Studies of thermophilic microorganisms have shown that they have a considerable biotechnological potential due to their optimum growth and metabolism at high temperatures. Thermophilic archaea have unique characteristics with important biotechnological applications; many of these species could be used in bioleaching processes to recover valuable metals from mineral ores. Particularly, bioleaching at high temperatures using thermoacidophilic microorganisms can greatly improve metal solubilization from refractory mineral species such as chalcopyrite (CuFeS2), one of the most abundant and widespread copper-bearing minerals. Interfacial processes such as early cell adhesion, biofilm development, and the formation of passive layers on the mineral surface play important roles in the initial steps of bioleaching processes. The present work focused on the investigation of different bioleaching conditions using the thermoacidophilic archaeon Acidianus copahuensis DSM 29038 to elucidate which steps are pivotal during the chalcopyrite bioleaching. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) were used to visualize the microorganism–mineral interaction. Results showed that up to 85% of copper recovery from chalcopyrite could be achieved using A. copahuensis. Improvements in these yields are intimately related to an early contact between cells and the mineral surface. On the other hand, surface coverage by inactivated cells as well as precipitates significantly reduced copper recoveries.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

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