Visualisation and biovolume quantification in the characterisation of biofilm formation in Mycoplasma fermentans

AbstractThe ability of mycoplasmas to persist on surfaces has been widely acknowledged, despite their fastidious nature. However, the organism’s capability to form a recognisable biofilm structure has been identified more recently. In the current study Mycoplasma fermentans was found to adhere to the glass surface forming highly differentiated biofilm structures. The volumes of biofilm microcolonies were quantified and observed to be greater at late growth stage than those at early growth stage. The channel diameters within biofilms were measured with Scanning Electron Microscopy images and found to be consistent with the size observed in Confocal Laser Scanning Microscope images. The combination of imaging methods with 3D visualisation provides key findings that aid understanding of the mycoplasma biofilm formation and true biofilm architecture. The observations reported here provide better understanding of the persistence of these minimalist pathogens in nature and clinical settings.

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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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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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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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Bacterial Growth on Chitosan-Coated Polypropylene Textile

ISRN Microbiology ◽

10.5402/2012/749694 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

D. Erben ◽

V. Hola ◽

J. Jaros ◽

J. Rahel

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Antibacterial Properties ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Aqueous Environment ◽

Bacterial Enumeration ◽

Pressure Development ◽

Barrier Discharge Plasma ◽

Biofilm Structure

Biofouling is a problem common in all systems where microorganisms and aqueous environment meet. Prevention of biofouling is therefore important in many industrial processes. The aim of this study was to develop a method to evaluate the ability of material coating to inhibit biofilm formation. Chitosan-coated polypropylene nonwoven textile was prepared using dielectric barrier discharge plasma activation. Resistance of the textile to biofouling was then tested. First, the textile was submerged into a growth medium inoculated with green fluorescein protein labelled Pseudomonas aeruginosa. After overnight incubation at 33°C, the textile was observed using confocal laser scanning microscopy for bacterial enumeration and biofilm structure characterisation. In the second stage, the textile was used as a filter medium for prefiltered river water, and the pressure development on the in-flow side was measured to quantify the overall level of biofouling. In both cases, nontreated textile samples were used as a control. The results indicate that the chitosan coating exhibits antibacterial properties. The developed method is applicable for the evaluation of the ability to inhibit biofilm formation.

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Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

Journal of Bacteriology ◽

10.1128/jb.00246-06 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4474-4486 ◽

Cited By ~ 84

Author(s):

Daniela M. Russo ◽

Alan Williams ◽

Anne Edwards ◽

Diana M. Posadas ◽

Christine Finnie ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Rhizobium Leguminosarum ◽

Cellular Adhesion ◽

Secretion System ◽

Laser Scanning Microscopy ◽

Secreted Proteins ◽

Confocal Laser ◽

Type I ◽

Biofilm Structure

ABSTRACT The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.

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3-D imaging of cells using a confocal laser scanning microscope and digital image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102560 ◽

1988 ◽

Vol 46 ◽

pp. 96-97

Author(s):

Thomas M. Jovin ◽

Michel Robert-Nicoud ◽

Donna J. Arndt-Jovin ◽

Thorsten Schormann

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Scanning Microscope ◽

Laser Scanning Microscope ◽

Biochemical Processes ◽

Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168554 ◽

1994 ◽

Vol 52 ◽

pp. 164-165

Author(s):

Thomas J. Deerinck ◽

Maryann E. Martone ◽

Varda Lev-Ram ◽

David P. L. Green ◽

Roger Y. Tsien ◽

...

Keyword(s):

Laser Scanning ◽

Reactive Oxygen ◽

Confocal Laser Scanning Microscope ◽

Fluorescent Dye ◽

Confocal Laser ◽

3 Dimensional ◽

Voltage Electron ◽

Dimensional Distribution ◽

Electron Microscopes ◽

New Generation

The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

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A confocal laser scanning microscope for fluorescence and reflection at video rates

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152641 ◽

1989 ◽

Vol 47 ◽

pp. 134-135

Author(s):

P.M. Houpt ◽

A. Draaijer

Keyword(s):

Light Source ◽

Laser Scanning ◽

Focal Point ◽

Confocal Laser Scanning Microscope ◽

Confocal Laser ◽

Point Light Source ◽

Volume Of Interest ◽

Ion Laser ◽

Point Light ◽

Point Detector

In confocal microscopy, the object is scanned by the coinciding focal points (confocal) of a point light source and a point detector both focused on a certain plane in the object. Only light coming from the focal point is detected and, even more important, out-of-focus light is rejected.This makes it possible to slice up optically the ‘volume of interest’ in the object by moving it axially while scanning the focused point light source (X-Y) laterally. The successive confocal sections can be stored in a computer and used to reconstruct the object in a 3D image display.The instrument described is able to scan the object laterally with an Ar ion laser (488 nm) at video rates. The image of one confocal section of an object can be displayed within 40 milliseconds (1000 х 1000 pixels). The time to record the total information within the ‘volume of interest’ normally depends on the number of slices needed to cover it, but rarely exceeds a few seconds.

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Use of confocal laser scanning microscopy and a computer model to understand ink cavitation and filamentation

TAPPI Journal ◽

10.32964/tj9.10.7 ◽

2010 ◽

Vol 9 (10) ◽

pp. 7-15

Author(s):

HANNA KOIVULA ◽

DOUGLAS BOUSFIELD ◽

MARTTI TOIVAKKA

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Print Quality ◽

Length Scale ◽

Offset Printing ◽

Significant Difference ◽

Printing Speed

In the offset printing process, ink film splitting has an important impact on formation of ink filaments. The filament size and its distribution influence the leveling of ink and hence affect ink setting and the print quality. However, ink filaments are difficult to image due to their short lifetime and fine length scale. Due to this difficulty, limited work has been reported on the parameters that influence filament size and methods to characterize it. We imaged ink filament remains and quantified some of their characteristics by changing printing speed, ink amount, and fountain solution type. Printed samples were prepared using a laboratory printability tester with varying ink levels and operating settings. Rhodamine B dye was incorporated into fountain solutions to aid in the detection of the filaments. The prints were then imaged with a confocal laser scanning microscope (CLSM) and images were further analyzed for their surface topography. Modeling of the pressure pulses in the printing nip was included to better understand the mechanism of filament formation and the origin of filament length scale. Printing speed and ink amount changed the size distribution of the observed filament remains. There was no significant difference between fountain solutions with or without isopropyl alcohol on the observed patterns of the filament remains.

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Confocal and SEM imaging to demonstrate food pathogen- biofilm biocontrol by pyocyanin from Pseudomonas aeruginosa BTRY1

International Journal of Bioassays ◽

10.21746/ijbio.2017.01.007 ◽

2016 ◽

Vol 6 (01) ◽

pp. 5218

Author(s):

Laxmi Mohandas ◽

Anju T. R. ◽

Sarita G. Bhat*

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity ◽

Opportunistic Pathogens ◽

Redox Active ◽

Sem Imaging ◽

The Impact

An assortment of redox-active phenazine compounds like pyocyanin with their characteristic blue-green colour are synthesized by Pseudomonas aeruginosa, Gram-negative opportunistic pathogens, which are also considered one of the most commercially valuable microorganisms. In this study, pyocyanin from Pseudomonas aeruginosa BTRY1 from food sample was assessed for its antibiofilm activity by micro titer plate assay against strong biofilm producers belonging to the genera Bacillus, Staphylococcus, Brevibacterium and Micrococcus. Pyocyanin inhibited biofilm activity in very minute concentrations. This was also confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Both SEM and CLSM helped to visualize the biocontrol of biofilm formation by eight pathogens. The imaging and quantification by CLSM also established the impact of pyocyanin on biofilm-biocontrol mainly in the food industry.

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