Combined application of 13C NMR spectroscopy and confocal laser scanning microscopy—Investigation on biofilm structure and physico-chemical properties

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.

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Automated biofilm morphology quantification from confocal laser scanning microscopy imaging

Water Science & Technology ◽

10.2166/wst.2003.0273 ◽

2003 ◽

Vol 47 (5) ◽

pp. 31-37 ◽

Cited By ~ 47

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.

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Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy

Vascular ◽

10.1177/1708538120917555 ◽

2020 ◽

Vol 28 (5) ◽

pp. 619-628

Author(s):

Xiao-Ling Zhuang ◽

Zhuang-Li Zhu ◽

Jie-Ling Zhu ◽

Su-Mei Lai ◽

Long-Xin Gui ◽

...

Keyword(s):

Nitric Oxide ◽

Pulmonary Artery ◽

Confocal Laser Scanning Microscopy ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Combined Application ◽

M 10 ◽

No Release

Objectives To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. Methods In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100–300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10−6 M), and subsequently dilated in acetylcholine (ACh, 10−6 M – 10−4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10−4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10−6 M) followed by ACh (10−5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. Results ACh (10−6 M – 10−4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10−4 M) significantly attenuated the vasodilatation and the intracellular NO release. Conclusions This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

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

Water Science & Technology ◽

10.2166/wst.2004.0834 ◽

2004 ◽

Vol 49 (11-12) ◽

pp. 177-185 ◽

Cited By ~ 24

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.

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Sorption and metabolism of selected herbicides in river biofilm communities

Canadian Journal of Microbiology ◽

10.1139/w01-061 ◽

2001 ◽

Vol 47 (7) ◽

pp. 634-641 ◽

Cited By ~ 56

Author(s):

J R Lawrence ◽

G Kopf ◽

J V Headley ◽

T R Neu

Keyword(s):

Laser Scanning ◽

Maximum Level ◽

Sole Source ◽

Bulk Water ◽

Laser Scanning Microscopy ◽

Water Phase ◽

Confocal Laser ◽

Community Members ◽

River Biofilm ◽

Biofilm Structure

In the present study, biofilms were grown in rotating annular bioreactors with river water as inoculum and sole source of nutrients. The herbicides atrazine and diclofop methyl were applied to the bioreactors, while an identical reactor acted as a control. Biofilm structure was visualized using specific fluorescent probes in conjunction with confocal laser scanning microscopy. The concentration of both herbicides in the bulk water phase followed the pattern of application. Atrazine and metabolites were detected in biofilm samples using direct insertion probe tandem mass spectrometry (DIP-MS/MS) and only trace levels were detected after the addition phase. Monoclonal antibody (MAb) studies indicated that sorption of atrazine was associated with a unique microcolony type. In contrast, diclofop and metabolites reached a maximum level in the biofilm at the end of the addition phase and persisted in the biofilm. Experiments with14C-labeled atrazine and diclofop methyl indicated that mineralization of these compounds to CO2(<1%) occurred in the river biofilms. Thus, both herbicides were sorbed and metabolized by the river biofilm community and detected in biofilms when they were not detected in the bulk water phase. These results indicate that biofilms and specific community members may act as a sink for herbicides, and that this should be taken into account in terms of both sampling and studies of the environmental chemodynamics of contaminants.Key words: biofilm, confocal, image analyses, herbicide, sorption.

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Nicotine Enhances Interspecies Relationship betweenStreptococcus mutansandCandida albicans

BioMed Research International ◽

10.1155/2017/7953920 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Shiyu Liu ◽

Wei Qiu ◽

Keke Zhang ◽

Xuedong Zhou ◽

Biao Ren ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Extracellular Polysaccharides ◽

Confocal Laser ◽

Fungal Cell ◽

Biofilm Model ◽

Structure Synthesis ◽

Biofilm Structure ◽

Synergistic Relationship

Streptococcus mutansandCandida albicansare common microorganisms in the human oral cavity. The synergistic relationship between these two species has been deeply explored in many studies. In the present study, the effect of alkaloid nicotine on the interspecies betweenS. mutansandC. albicansis explored. We developed a dual-species biofilm model and studied biofilm biomass, biofilm structure, synthesis of extracellular polysaccharides (EPS), and expression of glucosyltransferases (Gtfs). Biofilm formation and bacterial and fungal cell numbers in dual-species biofilms increased in the presence of nicotine. MoreC. albicanscells were present in the dual-species biofilms in the nicotine-treated groups as determined by scanning electron microscopy. The synthesis of EPS was increased by 1 mg/ml of nicotine as detected by confocal laser scanning microscopy. The result of qRT-PCR showedgtfsexpression was upregulated when 1 mg/ml of nicotine was used. We speculate that nicotine promoted the growth ofS. mutans, and moreS. mutanscells attracted moreC. albicanscells due to the interaction between two species. SinceS. mutansandC. albicansare putative pathogens for dental caries, the enhancement of the synergistic relationship by nicotine may contribute to caries development in smokers.

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Advances in mathematical modeling of biofilm structure

Biofilms ◽

10.1017/s1479050505001572 ◽

2004 ◽

Vol 1 (4) ◽

pp. 337-349 ◽

Cited By ~ 34

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.

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Experimental Assessment of the Performance of Two Marine Coatings to Curb Biofilm Formation of Microfoulers

Coatings ◽

10.3390/coatings10090893 ◽

2020 ◽

Vol 10 (9) ◽

pp. 893 ◽

Cited By ~ 1

Author(s):

Sara I. Faria ◽

Rita Teixeira-Santos ◽

Luciana C. Gomes ◽

Elisabete R. Silva ◽

João Morais ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Critical Role ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Marine Biofouling ◽

Marine Coatings ◽

Antifouling Coatings ◽

Wet Weight ◽

Biofilm Structure

Biofilms formed on submerged marine surfaces play a critical role in the fouling process, causing increased fuel consumption, corrosion, and high maintenance costs. Thus, marine biofouling is a major issue and motivates the development of antifouling coatings. In this study, the performance of two commercial marine coatings, a foul-release silicone-based paint (SilRef) and an epoxy resin (EpoRef), was evaluated regarding their abilities to prevent biofilm formation by Cyanobium sp. and Pseudoalteromonas tunicata (common microfoulers). Biofilms were developed under defined hydrodynamic conditions to simulate marine settings, and the number of biofilm cells, wet weight, and thickness were monitored for 7 weeks. The biofilm structure was analyzed by confocal laser scanning microscopy (CLSM) at the end-point. Results demonstrated that EpoRef surfaces were effective in inhibiting biofilm formation at initial stages (until day 28), while SilRef surfaces showed high efficacy in decreasing biofilm formation during maturation (from day 35 onwards). Wet weight and thickness analysis, as well as CLSM data, indicate that SilRef surfaces were less prone to biofilm formation than EpoRef surfaces. Furthermore, the efficacy of SilRef surfaces may be dependent on the fouling microorganism, while the performance of EpoRef was strongly influenced by a combined effect of surface and microorganism.

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Physico-chemical characterisation versus in situ micro-structural characterisation of membrane fouling in membrane bioreactors

Water Science & Technology ◽

10.2166/wst.2011.196 ◽

2011 ◽

Vol 63 (8) ◽

pp. 1781-1787 ◽

Cited By ~ 2

Author(s):

T. C. A Ng ◽

H. Y. Ng

Keyword(s):

Membrane Fouling ◽

Laser Scanning ◽

Extracellular Polymeric Substances ◽

Membrane Bioreactors ◽

Operating Conditions ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Critical Flux ◽

Membrane Materials ◽

Physico Chemical

Fouling characteristics of aerobic submerged membrane bioreactors were analysed under two different membrane materials. Polyethersulfone (PES) membranes were found to foul faster at sub-critical flux than polyolefin (PO) membranes. Physico-chemical characterisation, by means of comparison of extracellular polymeric substances (EPS) and soluble microbial products (SMP) concentrations, as well as the mixed liquor suspended solids (MLSS) concentration were unable to explain the differences in membrane fouling of the contrasting membrane materials. The use of confocal laser scanning microscopy (CLSM) to image organic foulants directly on the membrane surface, coupled with image analyses showed that membrane fouling mechanism shifted from a biofilm initiated process on PO membranes to a bio-organic dominated process on PES membranes under sub-critical flux conditions. These results show that physico-chemical characterisation of an MBR process may not effectively distinguish the effectiveness of different membrane materials, so long as operating conditions are identical, and that characterisation of foulants on the membrane surfaces was necessary to elucidate the differences in membrane fouling.

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Cold Shock Fail to Restrain Pre-formed Bacterial Biofilm

10.1101/324749 ◽

2018 ◽

Author(s):

Wenying Yu ◽

Qiao Han ◽

Xueying Song ◽

Jiaojiao Fu ◽

Haiquan Liu ◽

...

Keyword(s):

Correlation Analysis ◽

Biofilm Formation ◽

Laser Scanning ◽

Cold Shock ◽

Bacterial Biofilm ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Structure Parameters ◽

Cold Environment ◽

Biofilm Structure

ABSTRACTEnvironmental temperature fluctuation has great impact on the formation of bacterial biofilm, while little information is available for assessing the influence of sharp temperature shifts on the fate of pre-formed biofilm. In this study, experimental evidence is firstly explored on the response ofVibrio parahaemolyticuspre-formed biofilm under cold shock (4 °C and 10 °C). Surprisingly, biofilm biomass ofV. parahaemolyticussignificantly increased during the period of cold shock as revealed by crystal violet staining. Polysaccharides and proteins contents in extracellular polymeric substances were gradually enhanced after cold shocks and exhibited high consistency. RT-qPCR demonstrated the expression of flagella and virulence-related genes were up-regulated. Most of QS and T3SS genes were slightly up-regulated, and three T3SS genes (vcrD1,vcrD2βandvopD1) were down-regulated. Furthermore, the biofilm structure ofV parahaemolyticushave been analyzed by Confocal laser scanning microscopy (CLSM), which sharply changed under cold shocks. The correlation analysis further displayed the significant correlation (P < 0.01) among biofilm structure parameters, and weak correlation (P < 0.05) between biofilm related genes and biofilm structure parameters. In conclusion, our results novel discovered thatV. parahaemolyticusbiofilm related genes were actively expressed and biofilm biomass was continuously increased, biofilm structure was tremendously changed after cold shock. This study underscored the risk that biofilm cells had the ability to adapt to low temperature shift.IMPORTANCEBiofilms are widespread in natural environments, especially on the surface of food and medical biomaterials, which threaten human safety from persistent infections. Previous studies simply focused on biofilm formation of microorganisms under steady state, however, the actual environment frequently fluctuated.V. parahaemolyticusis a widely distributed foodborne pathogen, temperature play a great role in its survival. Researchers generally assume that cold environment can restrain biofilm formation and bacterial activity. This study explored the effects ofV. parahaemolyticusbiofilm upon a shift from 37 °C to 4 °C or 10 °C from two aspects. On the one hand, the changes of biofilm biomass and EPS contents, the expression of biofilm related genes directly described that pre-formed bacterial biofilm could not be controlled efficiently in cold environment. On the other hand, the CLSM images revealed biofilm morphological structure change, the correlation analysis showed inner relationship among biofilm structure parameters and biofilm related genes. These results suggested that cold shock fail to restrain pre-formed bacterial biofilm, therefore be a potential risk in nature environment.

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