Structured Review

Bitplane biofilms
Characteristics of P. aeruginosa wild-type (filled circles) and mucA 22 mutant (open circles) <t>biofilms.</t> The biomass content, biofilm thickness, roughness coefficient, and substratum coverage were calculated by the COMSTAT image analysis software from scanning confocal image data. The values are averages of six image stacks acquired in four separate experiments. The biomass content is calculated as the biomass volume (μm 3 ) per substratum surface area (μm 2 ). The roughness coefficient describes the variation in biofilm thickness and is a measure of biofilm heterogeneity. The substratum coverage is the fraction of the substratum area covered by biomass. The times at which the biofilms were assayed were 1, 2, 4, 6, and 8 days (as indicated on the x axis).
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Images

1) Product Images from "Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function"

Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.18.5395-5401.2001

Characteristics of P. aeruginosa wild-type (filled circles) and mucA 22 mutant (open circles) biofilms. The biomass content, biofilm thickness, roughness coefficient, and substratum coverage were calculated by the COMSTAT image analysis software from scanning confocal image data. The values are averages of six image stacks acquired in four separate experiments. The biomass content is calculated as the biomass volume (μm 3 ) per substratum surface area (μm 2 ). The roughness coefficient describes the variation in biofilm thickness and is a measure of biofilm heterogeneity. The substratum coverage is the fraction of the substratum area covered by biomass. The times at which the biofilms were assayed were 1, 2, 4, 6, and 8 days (as indicated on the x axis).
Figure Legend Snippet: Characteristics of P. aeruginosa wild-type (filled circles) and mucA 22 mutant (open circles) biofilms. The biomass content, biofilm thickness, roughness coefficient, and substratum coverage were calculated by the COMSTAT image analysis software from scanning confocal image data. The values are averages of six image stacks acquired in four separate experiments. The biomass content is calculated as the biomass volume (μm 3 ) per substratum surface area (μm 2 ). The roughness coefficient describes the variation in biofilm thickness and is a measure of biofilm heterogeneity. The substratum coverage is the fraction of the substratum area covered by biomass. The times at which the biofilms were assayed were 1, 2, 4, 6, and 8 days (as indicated on the x axis).

Techniques Used: Mutagenesis, Software

Biofilm formation assay comparing the total biofilm biomasses of PAO1, PDO300, and algDmucA strains after 10 h. Biofilms were prepared and stained as described in Materials and Methods. Both PDO300 and the algDmucA double mutant harbored less biomass in their biofilms than did PAO1. Each value was the average of 32 individual replicates. Avg, average; St. dev., standard deviation.
Figure Legend Snippet: Biofilm formation assay comparing the total biofilm biomasses of PAO1, PDO300, and algDmucA strains after 10 h. Biofilms were prepared and stained as described in Materials and Methods. Both PDO300 and the algDmucA double mutant harbored less biomass in their biofilms than did PAO1. Each value was the average of 32 individual replicates. Avg, average; St. dev., standard deviation.

Techniques Used: Tube Formation Assay, Staining, Mutagenesis, Standard Deviation

Epifluorescence and scanning confocal photomicrographs of the surface-attached communities formed by P. aeruginosa wild type and an isogenic alginate-overproducing mutant. The strains are engineered to contain a gfp expression cassette inserted into the chromosome. The biofilms are grown in once flow-through continuous-culture reaction vessels. (Top) Epifluorescence photomicrographs of the wild type (PAO1) and the mucA 22 mutant (PDO300); images were acquired 8 h postinoculation of the biofilm reactor. (Middle) Epifluorescence photomicrographs acquired 24 h postinoculation. (Bottom) Scanning confocal photomicrographs of 5-day-old wild-type and mucA 22 mutant biofilms. The larger central plots are simulated fluorescent projections, in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. Bar, 20 μm.
Figure Legend Snippet: Epifluorescence and scanning confocal photomicrographs of the surface-attached communities formed by P. aeruginosa wild type and an isogenic alginate-overproducing mutant. The strains are engineered to contain a gfp expression cassette inserted into the chromosome. The biofilms are grown in once flow-through continuous-culture reaction vessels. (Top) Epifluorescence photomicrographs of the wild type (PAO1) and the mucA 22 mutant (PDO300); images were acquired 8 h postinoculation of the biofilm reactor. (Middle) Epifluorescence photomicrographs acquired 24 h postinoculation. (Bottom) Scanning confocal photomicrographs of 5-day-old wild-type and mucA 22 mutant biofilms. The larger central plots are simulated fluorescent projections, in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. Bar, 20 μm.

Techniques Used: Mutagenesis, Expressing, Flow Cytometry

Increased tobramycin resistance of a P. aeruginosa PDO300 biofilm. (A) Viable biomass content of gfp-expressing P. aeruginosa wild-type and PDO300 mutant biofilms after 24 h of exposure to 2.0 μg of tobramycin per ml (open bars) and nontreated controls (filled bars). gfp fluorescence is a marker of cell viability and allows quantification of the viable biomass by COMSTAT image analysis of SCLM image data. (B) Visualization of live (green fluorescence) and dead (red fluorescence) cells by LIVE/DEAD Bac Light bacterial viability staining kit. The treated biofilms were exposed to tobramycin as described above. Viability was measured by GFP fluorescence (A) and by SYTO 9 viability staining (B).
Figure Legend Snippet: Increased tobramycin resistance of a P. aeruginosa PDO300 biofilm. (A) Viable biomass content of gfp-expressing P. aeruginosa wild-type and PDO300 mutant biofilms after 24 h of exposure to 2.0 μg of tobramycin per ml (open bars) and nontreated controls (filled bars). gfp fluorescence is a marker of cell viability and allows quantification of the viable biomass by COMSTAT image analysis of SCLM image data. (B) Visualization of live (green fluorescence) and dead (red fluorescence) cells by LIVE/DEAD Bac Light bacterial viability staining kit. The treated biofilms were exposed to tobramycin as described above. Viability was measured by GFP fluorescence (A) and by SYTO 9 viability staining (B).

Techniques Used: Expressing, Mutagenesis, Fluorescence, Marker, BAC Assay, Staining

Assay of tobramycin sensitivity in a rotating-disk biofilm reactor system. ○, P. aeruginosa wild-type biofilm; ●, PDO300 mutant biofilm. The values represent averages of three separate experiments. Biofilms were treated for 5 h. The planktonic MIC of tobramycin for both these strains is 1 μg/ml.
Figure Legend Snippet: Assay of tobramycin sensitivity in a rotating-disk biofilm reactor system. ○, P. aeruginosa wild-type biofilm; ●, PDO300 mutant biofilm. The values represent averages of three separate experiments. Biofilms were treated for 5 h. The planktonic MIC of tobramycin for both these strains is 1 μg/ml.

Techniques Used: Mutagenesis

2) Product Images from "Identification and Characterization of OscR, a Transcriptional Regulator Involved in Osmolarity Adaptation in Vibrio cholerae ▿ ▿ †"

Article Title: Identification and Characterization of OscR, a Transcriptional Regulator Involved in Osmolarity Adaptation in Vibrio cholerae ▿ ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01540-08

(v) Salinity modulates biofilm formation through VpsR and VpsT.
Figure Legend Snippet: (v) Salinity modulates biofilm formation through VpsR and VpsT.

Techniques Used:

(iii) Biofilms are modulated by salinity.
Figure Legend Snippet: (iii) Biofilms are modulated by salinity.

Techniques Used:

Model of salinity/osmolarity response in V. cholerae . Under low-salinity conditions, oscR is upregulated, which in turn positively regulates motility and negatively regulates biofilm formation. At a median salinity, biofilm matrix gene expression is increased
Figure Legend Snippet: Model of salinity/osmolarity response in V. cholerae . Under low-salinity conditions, oscR is upregulated, which in turn positively regulates motility and negatively regulates biofilm formation. At a median salinity, biofilm matrix gene expression is increased

Techniques Used: Expressing

Osmolarity affects vps expression and biofilm architecture. A comparison of vpsL expression was performed using β-galactosidase assays of a strain harboring a chromosomal vpsLp-lacZ reporter grown to mid-exponential growth phase in NaCl (A), lactose
Figure Legend Snippet: Osmolarity affects vps expression and biofilm architecture. A comparison of vpsL expression was performed using β-galactosidase assays of a strain harboring a chromosomal vpsLp-lacZ reporter grown to mid-exponential growth phase in NaCl (A), lactose

Techniques Used: Expressing

(v) OscR modulates biofilm formation under low-salt conditions only.
Figure Legend Snippet: (v) OscR modulates biofilm formation under low-salt conditions only.

Techniques Used:

Salinity modulates vpsL expression and biofilm formation through VpsT and VpsR. (A) qPCR analysis of gyrA , vpsR , and vpsT message levels in wild-type cells grown to mid-exponential phase in LB supplemented with NaCl at the indicated concentrations. Gel
Figure Legend Snippet: Salinity modulates vpsL expression and biofilm formation through VpsT and VpsR. (A) qPCR analysis of gyrA , vpsR , and vpsT message levels in wild-type cells grown to mid-exponential phase in LB supplemented with NaCl at the indicated concentrations. Gel

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

3) Product Images from "The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans"

Article Title: The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.00687

The influences of rnc gene on S. mutans cariogenicity in the SPF rat model. (A) Representative three-dimensional images of biofilms that formed on occlusal plane of rat mandibular molars in four different groups by CLSM. Images were taken at 63× magnification. (B) SEM observation of the architecture of biofilms that formed on occlusal plane of rat mandibular molars. Images were taken at 20,000× magnification.
Figure Legend Snippet: The influences of rnc gene on S. mutans cariogenicity in the SPF rat model. (A) Representative three-dimensional images of biofilms that formed on occlusal plane of rat mandibular molars in four different groups by CLSM. Images were taken at 63× magnification. (B) SEM observation of the architecture of biofilms that formed on occlusal plane of rat mandibular molars. Images were taken at 20,000× magnification.

Techniques Used: Confocal Laser Scanning Microscopy

The effects of rnc gene on exopolysaccharide synthesis and biofilm morphology in S. mutans . The anthrone assay was used to determine glucan productions in UA159, Smurnc, and Smurnc + biofilms at 24 h. WSGs (A) and WIGs (B) were both calculated. The Smurnc strain decreased the amount of glucans, whereas the Smurnc + strain increased the amount. The error bars represent standard deviation values. Asterisks indicate significant differences ( P
Figure Legend Snippet: The effects of rnc gene on exopolysaccharide synthesis and biofilm morphology in S. mutans . The anthrone assay was used to determine glucan productions in UA159, Smurnc, and Smurnc + biofilms at 24 h. WSGs (A) and WIGs (B) were both calculated. The Smurnc strain decreased the amount of glucans, whereas the Smurnc + strain increased the amount. The error bars represent standard deviation values. Asterisks indicate significant differences ( P

Techniques Used: Standard Deviation

4) Product Images from "The TibA Adhesin/Invasin from Enterotoxigenic Escherichia coli Is Self Recognizing and Induces Bacterial Aggregation and Biofilm Formation "

Article Title: The TibA Adhesin/Invasin from Enterotoxigenic Escherichia coli Is Self Recognizing and Induces Bacterial Aggregation and Biofilm Formation

Journal: Infection and Immunity

doi: 10.1128/IAI.73.4.1954-1963.2005

Spatial distribution of biofilm formation for gfp -labeled E. coli MS427 derivative strains. (A) pACYC184 (vector control) (strain OS70). (B) pRMV4b (TibCA + ) (strain OS136). (C) pRMV1 (TibA + ) (strain RMV3). Biofilm development was monitored
Figure Legend Snippet: Spatial distribution of biofilm formation for gfp -labeled E. coli MS427 derivative strains. (A) pACYC184 (vector control) (strain OS70). (B) pRMV4b (TibCA + ) (strain OS136). (C) pRMV1 (TibA + ) (strain RMV3). Biofilm development was monitored

Techniques Used: Labeling, Plasmid Preparation

5) Product Images from "Prevalence, Molecular Typing, and Determination of the Biofilm-Forming Ability of Listeria monocytogenes Serotypes from Poultry Meat and Poultry Preparations in Spain"

Article Title: Prevalence, Molecular Typing, and Determination of the Biofilm-Forming Ability of Listeria monocytogenes Serotypes from Poultry Meat and Poultry Preparations in Spain

Journal: Microorganisms

doi: 10.3390/microorganisms7110529

Tree diagrams showing grouping of values for quantitative parameters of biofilms. These are biovolume ( A ) percentage of surface covered ( B ) roughness ( C ) and maximum thickness ( D ) corresponding to 20 strains of Listeria monocytogenes taken from meat (Euclidean distance, unweighted pair-group average).
Figure Legend Snippet: Tree diagrams showing grouping of values for quantitative parameters of biofilms. These are biovolume ( A ) percentage of surface covered ( B ) roughness ( C ) and maximum thickness ( D ) corresponding to 20 strains of Listeria monocytogenes taken from meat (Euclidean distance, unweighted pair-group average).

Techniques Used:

Three-dimensional projections of structures of biofilms obtained from one-micron optical sections on the z -axis acquired through confocal laser scanning microscopy. These images represent an overhead view of the biofilms formed by 20 strains of Listeria monocytogenes , with virtual projection of shadows to the right. Each square represented had length of side of 119 μm. Strains: 1 (serotype 1/2a), 2 (1/2a), 3 (1/2b), 4 (1/2b), 5 (1/2c), 6 (1/2c), 7 (3a), 8 (3b), 9 (3b), 10 (3c), 11 (3c), 12 (4a), 13 (4a), 14 (4b), 15 (4b), 16 (4b), 17 (4b), 18 (4c), 19 (4d), 20 (4d).
Figure Legend Snippet: Three-dimensional projections of structures of biofilms obtained from one-micron optical sections on the z -axis acquired through confocal laser scanning microscopy. These images represent an overhead view of the biofilms formed by 20 strains of Listeria monocytogenes , with virtual projection of shadows to the right. Each square represented had length of side of 119 μm. Strains: 1 (serotype 1/2a), 2 (1/2a), 3 (1/2b), 4 (1/2b), 5 (1/2c), 6 (1/2c), 7 (3a), 8 (3b), 9 (3b), 10 (3c), 11 (3c), 12 (4a), 13 (4a), 14 (4b), 15 (4b), 16 (4b), 17 (4b), 18 (4c), 19 (4d), 20 (4d).

Techniques Used: Confocal Laser Scanning Microscopy

6) Product Images from "Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans"

Article Title: Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans

Journal: Journal of Oral Microbiology

doi: 10.1080/20002297.2018.1442089

Effects of GH12 on polysaccharides synthesis and biofilm of S. mutans . (a) Three-dimensional CLSM image S. mutans biofilm (bacteria, stained green; EPS, stained red). (b) Vertical distribution of bacteria and EPS calculated from CLSM imaging data sets. (c) The biomass of EPS and bacteria, calculated according to five random sights of biofilms by COMSTATA. (d) Quantitative data of the water-insoluble EPS amount of S. mutans biofilms measured by the anthrone method. (e) Quantitative data of the S. mutans biofilm formation measured by crystal violet dye. Data are presented as means ± standard deviations. * P
Figure Legend Snippet: Effects of GH12 on polysaccharides synthesis and biofilm of S. mutans . (a) Three-dimensional CLSM image S. mutans biofilm (bacteria, stained green; EPS, stained red). (b) Vertical distribution of bacteria and EPS calculated from CLSM imaging data sets. (c) The biomass of EPS and bacteria, calculated according to five random sights of biofilms by COMSTATA. (d) Quantitative data of the water-insoluble EPS amount of S. mutans biofilms measured by the anthrone method. (e) Quantitative data of the S. mutans biofilm formation measured by crystal violet dye. Data are presented as means ± standard deviations. * P

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Imaging

7) Product Images from "Antimicrobial nisin acts against saliva derived multi-species biofilms without cytotoxicity to human oral cells"

Article Title: Antimicrobial nisin acts against saliva derived multi-species biofilms without cytotoxicity to human oral cells

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00617

Nisin inhibits the formation of multi-species biofilms in a Bioflux controlled flow microfluidic model system. CCS was added, then fed filter sterilized CFS for 20–22 h at 37°C with or without nisin. (A) Confocal microscopy images are represented in the x–y plane. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate channels (experiments). ∗ P
Figure Legend Snippet: Nisin inhibits the formation of multi-species biofilms in a Bioflux controlled flow microfluidic model system. CCS was added, then fed filter sterilized CFS for 20–22 h at 37°C with or without nisin. (A) Confocal microscopy images are represented in the x–y plane. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate channels (experiments). ∗ P

Techniques Used: Flow Cytometry, Confocal Microscopy, Derivative Assay, Imaging

Nisin inhibits the formation of multi-species biofilms in a static model system. Cell-containing saliva (CCS) was inoculated in filter sterilized cell-free saliva (CFS) for 20–22 h at 37°C with or without nisin. (A) Confocal microscopy images are represented in the x–y plane. A green signal indicates viable live cells (Syto 9), a red signal indicates damaged/dead cells (propidium iodide), (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate wells (experiments), (C) DNA content of the biofilms was quantified by absorption spectroscopy at fluorescence intensity of 530 nm. ∗ P
Figure Legend Snippet: Nisin inhibits the formation of multi-species biofilms in a static model system. Cell-containing saliva (CCS) was inoculated in filter sterilized cell-free saliva (CFS) for 20–22 h at 37°C with or without nisin. (A) Confocal microscopy images are represented in the x–y plane. A green signal indicates viable live cells (Syto 9), a red signal indicates damaged/dead cells (propidium iodide), (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate wells (experiments), (C) DNA content of the biofilms was quantified by absorption spectroscopy at fluorescence intensity of 530 nm. ∗ P

Techniques Used: Confocal Microscopy, Derivative Assay, Imaging, Spectroscopy, Fluorescence

Nisin disrupts the maintenance of three-dimensional architecture of pre-formed biofilms. CCS was inoculated in filter sterilized CFS for 20–22 h at 37°C and treated with phosphate buffered saline solution (PBS) solution (control) or nisin at different concentrations and incubation times. (A) Confocal microscopy images are represented in the x–y and x–y–z plane. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate wells (experiments). (C) An average percentage signal from the biofilms was determined by the Live/viable signal (green) and the Dead/damaged signal (red) in relation to the total signal captured for both. ∗ P
Figure Legend Snippet: Nisin disrupts the maintenance of three-dimensional architecture of pre-formed biofilms. CCS was inoculated in filter sterilized CFS for 20–22 h at 37°C and treated with phosphate buffered saline solution (PBS) solution (control) or nisin at different concentrations and incubation times. (A) Confocal microscopy images are represented in the x–y and x–y–z plane. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate wells (experiments). (C) An average percentage signal from the biofilms was determined by the Live/viable signal (green) and the Dead/damaged signal (red) in relation to the total signal captured for both. ∗ P

Techniques Used: Incubation, Confocal Microscopy, Derivative Assay, Imaging

8) Product Images from "EmbRS a new two-component system that inhibits biofilm formation and saves Rubrivivax gelatinosus from sinking"

Article Title: EmbRS a new two-component system that inhibits biofilm formation and saves Rubrivivax gelatinosus from sinking

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.82

Biofilm formation by ΔEmbRS cells. Formation of bacterial veils around the toothpick scaffolds. Cells were inoculated in the liquid medium and grown photosynthetically in plates. The extracellular matrix polymerization and its anchorage to the toothpicks led to the formation of the polygonal networks. (See movie M2).
Figure Legend Snippet: Biofilm formation by ΔEmbRS cells. Formation of bacterial veils around the toothpick scaffolds. Cells were inoculated in the liquid medium and grown photosynthetically in plates. The extracellular matrix polymerization and its anchorage to the toothpicks led to the formation of the polygonal networks. (See movie M2).

Techniques Used:

Biofilm formation by ΔEmbRS cells. (A) Digital camera images show macroscopic biofilm structures only in the ΔEmbRS mutant in microplates. (B) Microscopic confocal laser scanning microscopy (CLSM) visualization of the microtiter wells. 12-h biofilms were fluorescently labeled in green with syto 9. (C) Fluorescent (lectin ConA, red) labeling demonstrates the presence of higher amount of exopolysaccharide pockets in the 12-h biofilm of ΔEmbRS mutant compared to the WT.
Figure Legend Snippet: Biofilm formation by ΔEmbRS cells. (A) Digital camera images show macroscopic biofilm structures only in the ΔEmbRS mutant in microplates. (B) Microscopic confocal laser scanning microscopy (CLSM) visualization of the microtiter wells. 12-h biofilms were fluorescently labeled in green with syto 9. (C) Fluorescent (lectin ConA, red) labeling demonstrates the presence of higher amount of exopolysaccharide pockets in the 12-h biofilm of ΔEmbRS mutant compared to the WT.

Techniques Used: Mutagenesis, Confocal Laser Scanning Microscopy, Labeling

9) Product Images from "Bactericidal Compounds Controlling Growth of the Plant Pathogen Pseudomonas syringae pv. actinidiae, Which Forms Biofilms Composed of a Novel Exopolysaccharide"

Article Title: Bactericidal Compounds Controlling Growth of the Plant Pathogen Pseudomonas syringae pv. actinidiae, Which Forms Biofilms Composed of a Novel Exopolysaccharide

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00194-15

CLSM images of a P. syringae pv. actinidiae NZ V-13 biofilm treated with different concentrations of chlorine dioxide and normal saline in a flow cell system. (A) Normal saline-treated biofilm (control); (B to E) biofilms treated with 10 ppm (B), 20 ppm
Figure Legend Snippet: CLSM images of a P. syringae pv. actinidiae NZ V-13 biofilm treated with different concentrations of chlorine dioxide and normal saline in a flow cell system. (A) Normal saline-treated biofilm (control); (B to E) biofilms treated with 10 ppm (B), 20 ppm

Techniques Used: Confocal Laser Scanning Microscopy, Flow Cytometry

CLSM images of P. syringae pv. actinidiae NZ V-13 biofilm architecture in the flow cell system. (A and B) Top views of the biofilm structure, consisting of a base layer of cells scattered across the surface of the cover slide and microcolonies. (C and
Figure Legend Snippet: CLSM images of P. syringae pv. actinidiae NZ V-13 biofilm architecture in the flow cell system. (A and B) Top views of the biofilm structure, consisting of a base layer of cells scattered across the surface of the cover slide and microcolonies. (C and

Techniques Used: Confocal Laser Scanning Microscopy, Flow Cytometry

CLSM images of a P. syringae pv. actinidiae NZ V-13 biofilm treated with different concentrations of kasugamycin and normal saline in a flow cell system. (A) Normal saline-treated biofilm (control); (B to D) biofilms treated with 5 ppm (B) 10 ppm (C),
Figure Legend Snippet: CLSM images of a P. syringae pv. actinidiae NZ V-13 biofilm treated with different concentrations of kasugamycin and normal saline in a flow cell system. (A) Normal saline-treated biofilm (control); (B to D) biofilms treated with 5 ppm (B) 10 ppm (C),

Techniques Used: Confocal Laser Scanning Microscopy, Flow Cytometry

Ratios of live and dead cells of P. syringae pv. actinidiae NZ V-13 in biofilms treated with different concentrations of chlorine dioxide and kasugamycin grown in a flow cell system. (A) Results for P. syringae pv. actinidiae strain NZ V-13 treated with
Figure Legend Snippet: Ratios of live and dead cells of P. syringae pv. actinidiae NZ V-13 in biofilms treated with different concentrations of chlorine dioxide and kasugamycin grown in a flow cell system. (A) Results for P. syringae pv. actinidiae strain NZ V-13 treated with

Techniques Used: Flow Cytometry

Effect of different concentrations of kasugamycin on P. syringae pv. actinidiae NZ V-13 in the SSA assay. (A) Biofilm formation attachment phase ( T 0 ); (B) biofilms with maximum adherence treated with normal saline ( T max ); (C) effect of different concentrations
Figure Legend Snippet: Effect of different concentrations of kasugamycin on P. syringae pv. actinidiae NZ V-13 in the SSA assay. (A) Biofilm formation attachment phase ( T 0 ); (B) biofilms with maximum adherence treated with normal saline ( T max ); (C) effect of different concentrations

Techniques Used: SSA Assay

10) Product Images from "Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract ▿"

Article Title: Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.01952-06

(A) Schematic representation of the OxyR binding sites and adjacent nucleotide sequences of the flu gene from E. coli K-12 and the fluA and fluB genes from CFT073. The putative OxyR binding sites are underlined, the GATC methylation sites are indicated by bold type, and the nucleotide sequence differences are indicated by shading. (B) Ag43 expression in CFT073 flu and oxyR mutants. Protein preparations were obtained by subjecting cells to a brief heat treatment, centrifugation, and subsequent SDS-PAGE and immunodetection analysis using a serum directed against the α-domain of Ag43 from E. coli K-12. Whereas no Ag43 variants could be detected in wild-type CFT073 in this image, longer exposure of the same membrane revealed the presence of Ag43a but not Ag43b in CFT073 (data not shown). Deletion of oxyR resulted in significant expression of both Ag43a and Ag43b, demonstrating that OxyR represses both the fluA and fluB genes in wild-type strain CFT073. (C) Autoaggregation assay demonstrating the settling profiles for liquid suspensions of CFT073, CFT073 fluA , CFT073 fluB , CFT fluA fluB , CFT073 oxyR , CFT073 oxyR fluB , CFT073 oxyR fluA , and CFT073 oxyR fluA fluB . Deletion of oxyR did not produce an aggregation phenotype. (D) Biofilm formation by CFT073, CFT073 fluA , CFT073 fluB , CFT fluA fluB , CFT073 oxyR , CFT073 oxyR fluB , CFT073 oxyR fluA , and CFT073 oxyR fluA fluB . Deletion of oxyR gave rise to a biofilm phenotype similar to that of the CFT073 fluA + , CFT073 fluB + , and CFT073 fluA + fluB + strains.
Figure Legend Snippet: (A) Schematic representation of the OxyR binding sites and adjacent nucleotide sequences of the flu gene from E. coli K-12 and the fluA and fluB genes from CFT073. The putative OxyR binding sites are underlined, the GATC methylation sites are indicated by bold type, and the nucleotide sequence differences are indicated by shading. (B) Ag43 expression in CFT073 flu and oxyR mutants. Protein preparations were obtained by subjecting cells to a brief heat treatment, centrifugation, and subsequent SDS-PAGE and immunodetection analysis using a serum directed against the α-domain of Ag43 from E. coli K-12. Whereas no Ag43 variants could be detected in wild-type CFT073 in this image, longer exposure of the same membrane revealed the presence of Ag43a but not Ag43b in CFT073 (data not shown). Deletion of oxyR resulted in significant expression of both Ag43a and Ag43b, demonstrating that OxyR represses both the fluA and fluB genes in wild-type strain CFT073. (C) Autoaggregation assay demonstrating the settling profiles for liquid suspensions of CFT073, CFT073 fluA , CFT073 fluB , CFT fluA fluB , CFT073 oxyR , CFT073 oxyR fluB , CFT073 oxyR fluA , and CFT073 oxyR fluA fluB . Deletion of oxyR did not produce an aggregation phenotype. (D) Biofilm formation by CFT073, CFT073 fluA , CFT073 fluB , CFT fluA fluB , CFT073 oxyR , CFT073 oxyR fluB , CFT073 oxyR fluA , and CFT073 oxyR fluA fluB . Deletion of oxyR gave rise to a biofilm phenotype similar to that of the CFT073 fluA + , CFT073 fluB + , and CFT073 fluA + fluB + strains.

Techniques Used: Binding Assay, Methylation, Sequencing, Expressing, Centrifugation, SDS Page, Immunodetection

(A) Immunodetection analysis of Ag43a and Ag43b proteins produced in CFT073 as a result of constitutive expression from PcL promoter insertions. Protein preparations were obtained by subjecting cells to a brief heat treatment, centrifugation, and subsequent SDS-PAGE and immunodetection analysis using a serum directed against the α-domain of Ag43 from E. coli K-12. Each strain produced the appropriate Ag43 variant(s), as demonstrated by the different sizes of the alpha-domain and the degradation products (indicated by an asterisk). (B) Biofilm formation in microtiter plates by CFT073 expressing Ag43 variants and capsule and type 1 fimbria mutant derivatives. WT, wild type.
Figure Legend Snippet: (A) Immunodetection analysis of Ag43a and Ag43b proteins produced in CFT073 as a result of constitutive expression from PcL promoter insertions. Protein preparations were obtained by subjecting cells to a brief heat treatment, centrifugation, and subsequent SDS-PAGE and immunodetection analysis using a serum directed against the α-domain of Ag43 from E. coli K-12. Each strain produced the appropriate Ag43 variant(s), as demonstrated by the different sizes of the alpha-domain and the degradation products (indicated by an asterisk). (B) Biofilm formation in microtiter plates by CFT073 expressing Ag43 variants and capsule and type 1 fimbria mutant derivatives. WT, wild type.

Techniques Used: Immunodetection, Produced, Expressing, Centrifugation, SDS Page, Variant Assay, Mutagenesis

11) Product Images from "The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation"

Article Title: The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation

Journal: Journal of Bacteriology

doi: 10.1128/JB.00139-17

Analysis of biofilm gene expression in the wild type and in Δ vxrA and Δ vxrB mutants. (A) Cultures of wild-type, Δ vxrA , Δ vxrB , and Δ vxrB Tn 7 :: vxrB strains containing P vpsL - lux were grown to exponential phase (OD 600 of ∼0.3) and luminescence was measured. The graph represents the averages and standard deviations of RLU obtained from three technical replicates from three biological replicates. RLU are reported in luminescence counts · min −1 · ml −1 · OD 600 −1 . Data were analyzed using a one-way analysis of variance (ANOVA) and Bonferroni's multiple-comparison test. ***, P
Figure Legend Snippet: Analysis of biofilm gene expression in the wild type and in Δ vxrA and Δ vxrB mutants. (A) Cultures of wild-type, Δ vxrA , Δ vxrB , and Δ vxrB Tn 7 :: vxrB strains containing P vpsL - lux were grown to exponential phase (OD 600 of ∼0.3) and luminescence was measured. The graph represents the averages and standard deviations of RLU obtained from three technical replicates from three biological replicates. RLU are reported in luminescence counts · min −1 · ml −1 · OD 600 −1 . Data were analyzed using a one-way analysis of variance (ANOVA) and Bonferroni's multiple-comparison test. ***, P

Techniques Used: Expressing

Analysis of biofilm gene expression in the wild type and in response regulator (RR) deletion mutants. A transcriptional reporter harboring the regulatory region of vpsL upstream of a promoterless lux reporter (P vpsL - lux ) was used to analyze the expression of biofilm genes in 41 ΔRR mutants, including ΔVC0396 ( qstR ), ΔVC0665 ( vpsR ), ΔVC0693, ΔVC0719 ( phoB ), ΔVC0790, ΔVC1021 ( luxO ), ΔVC1050, ΔVC1081, ΔVC1082, ΔVC1086, ΔVC1087, ΔVC1155, ΔVC1213 ( varA ), ΔVC1277, ΔVC1320 ( carR ), ΔVC1348, ΔVC1522, ΔVC1604, ΔVC1638, ΔVC1651 ( vieB ), ΔVC1652 ( vieA ), ΔVC1719 ( torR ), ΔVC1926 ( dctD1 ), ΔVC2135 ( flrC ), ΔVC2137 ( flrA ), ΔVC2692 ( cpxR ), ΔVC2702 ( cbrR ), ΔVC2714 ( ompR ), ΔVC2749 ( ntrC ), ΔVCA0142 ( dctD2 ), ΔVCA0210, ΔVCA0239, ΔVCA0256, ΔVCA0532, ΔVCA0566 ( vxrB ), ΔVCA0682 ( uhpA ), ΔVCA0704 ( pgtA ), ΔVCA0850, ΔVCA0952 ( vpsT ), ΔVCA1086, and ΔVCA1105 strains (names included in parentheticals where relevant). Cultures of the wild type and ΔRR mutants were grown to exponential phase (OD 600 of ∼0.3) and luminescence was measured. The graph represents the averages and standard deviations of relative light units (RLU) obtained from at least three technical replicates from two biological replicates, normalized to wild-type levels. RLU are reported in luminescence counts · min −1 · ml −1 · OD 600 −1 . These values were then normalized to the wild-type RLU, and data are shown as fold changes above or below a wild-type value of 1. Data were analyzed using a one-way analysis of variance (ANOVA) and Bonferroni's multiple-comparison test. ***, P
Figure Legend Snippet: Analysis of biofilm gene expression in the wild type and in response regulator (RR) deletion mutants. A transcriptional reporter harboring the regulatory region of vpsL upstream of a promoterless lux reporter (P vpsL - lux ) was used to analyze the expression of biofilm genes in 41 ΔRR mutants, including ΔVC0396 ( qstR ), ΔVC0665 ( vpsR ), ΔVC0693, ΔVC0719 ( phoB ), ΔVC0790, ΔVC1021 ( luxO ), ΔVC1050, ΔVC1081, ΔVC1082, ΔVC1086, ΔVC1087, ΔVC1155, ΔVC1213 ( varA ), ΔVC1277, ΔVC1320 ( carR ), ΔVC1348, ΔVC1522, ΔVC1604, ΔVC1638, ΔVC1651 ( vieB ), ΔVC1652 ( vieA ), ΔVC1719 ( torR ), ΔVC1926 ( dctD1 ), ΔVC2135 ( flrC ), ΔVC2137 ( flrA ), ΔVC2692 ( cpxR ), ΔVC2702 ( cbrR ), ΔVC2714 ( ompR ), ΔVC2749 ( ntrC ), ΔVCA0142 ( dctD2 ), ΔVCA0210, ΔVCA0239, ΔVCA0256, ΔVCA0532, ΔVCA0566 ( vxrB ), ΔVCA0682 ( uhpA ), ΔVCA0704 ( pgtA ), ΔVCA0850, ΔVCA0952 ( vpsT ), ΔVCA1086, and ΔVCA1105 strains (names included in parentheticals where relevant). Cultures of the wild type and ΔRR mutants were grown to exponential phase (OD 600 of ∼0.3) and luminescence was measured. The graph represents the averages and standard deviations of relative light units (RLU) obtained from at least three technical replicates from two biological replicates, normalized to wild-type levels. RLU are reported in luminescence counts · min −1 · ml −1 · OD 600 −1 . These values were then normalized to the wild-type RLU, and data are shown as fold changes above or below a wild-type value of 1. Data were analyzed using a one-way analysis of variance (ANOVA) and Bonferroni's multiple-comparison test. ***, P

Techniques Used: Expressing

12) Product Images from "Growth of Pseudomonas taiwanensisVLB120∆C biofilms in the presence of n‐butanol"

Article Title: Growth of Pseudomonas taiwanensisVLB120∆C biofilms in the presence of n‐butanol

Journal: Microbial Biotechnology

doi: 10.1111/1751-7915.12413

Medium dependent biofilm growth of P. taiwanensis VLB 120∆C at increasing butanol concentrations. Data presented here are mean values from three different experiments.
Figure Legend Snippet: Medium dependent biofilm growth of P. taiwanensis VLB 120∆C at increasing butanol concentrations. Data presented here are mean values from three different experiments.

Techniques Used:

EPS profile of the biofilm grown with and without butanol. (A) Total amount of EPS produced by biofilms of P. taiwanensis VLB 120∆C under standard conditions and in the presence of butanol. (B) SEM images of the dehydrated biofilm grown without butanol. (C) With 0.5% butanol. (D) With 2% butanol for 10 days. White arrows show the cells and red arrows show the denatured EPS . (E and F) Main EPS compounds detected in the EPS of biofilms grown under standard conditions and in the presence of 0.5% butanol. Data presented here in (A, E and F) are mean value from four different experiments.
Figure Legend Snippet: EPS profile of the biofilm grown with and without butanol. (A) Total amount of EPS produced by biofilms of P. taiwanensis VLB 120∆C under standard conditions and in the presence of butanol. (B) SEM images of the dehydrated biofilm grown without butanol. (C) With 0.5% butanol. (D) With 2% butanol for 10 days. White arrows show the cells and red arrows show the denatured EPS . (E and F) Main EPS compounds detected in the EPS of biofilms grown under standard conditions and in the presence of 0.5% butanol. Data presented here in (A, E and F) are mean value from four different experiments.

Techniques Used: Produced

Cell size of P. taiwanensis VLB 120∆C biofilm grown with and without butanol. (A and B) SEM micrographs of the cellular morphology without butanol (A) and with 0.5% butanol (B). Arrows indicate the smaller cells. (C) Surface area‐to‐volume ratio of the cells grown at different butanol concentration for 2 and 10 days. Data presented here are mean value from two different experiments. The size of approximately 100 cells from each culture was measured.
Figure Legend Snippet: Cell size of P. taiwanensis VLB 120∆C biofilm grown with and without butanol. (A and B) SEM micrographs of the cellular morphology without butanol (A) and with 0.5% butanol (B). Arrows indicate the smaller cells. (C) Surface area‐to‐volume ratio of the cells grown at different butanol concentration for 2 and 10 days. Data presented here are mean value from two different experiments. The size of approximately 100 cells from each culture was measured.

Techniques Used: Concentration Assay

Physiological response and butanol concentration dependent biomass yield of P. taiwanensis VLB 120∆C biofilms. (A) Biofilm growth expressed as the amount of biomass produced on a given reactor surface in the presence of different butanol concentrations. (B) Cell numbers per square metre of the reactor surface. Biofilm was cultivated for 2 days. Data presented here are mean values from four parallel experiments.
Figure Legend Snippet: Physiological response and butanol concentration dependent biomass yield of P. taiwanensis VLB 120∆C biofilms. (A) Biofilm growth expressed as the amount of biomass produced on a given reactor surface in the presence of different butanol concentrations. (B) Cell numbers per square metre of the reactor surface. Biofilm was cultivated for 2 days. Data presented here are mean values from four parallel experiments.

Techniques Used: Concentration Assay, Produced

Micrographs showing biofilm development of P. taiwanensis VLB 120∆C egfp under standard growth conditions (no butanol) and in the presence of increasing butanol concentrations in minimal medium. Green colour represents the intact gfp ‐expressing cells; red colour represents PI ‐stained dead and/or permeabilized cells. Representative IMARIS ‐treated and 3D‐reconstructed images from three parallel experiments are shown. Scale bar, 20 μm.
Figure Legend Snippet: Micrographs showing biofilm development of P. taiwanensis VLB 120∆C egfp under standard growth conditions (no butanol) and in the presence of increasing butanol concentrations in minimal medium. Green colour represents the intact gfp ‐expressing cells; red colour represents PI ‐stained dead and/or permeabilized cells. Representative IMARIS ‐treated and 3D‐reconstructed images from three parallel experiments are shown. Scale bar, 20 μm.

Techniques Used: Expressing, Staining

13) Product Images from "Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis"

Article Title: Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis

Journal: Microbiology

doi: 10.1099/mic.0.040196-0

Biofilm structure analysis of a vps -clusters deletion mutant. CSLM images of horizontal ( xy ) and vertical ( xz ) projections (large and side panels, respectively) of biofilm structures formed by rugose wild-type and a mutant strain unable to produce VPS (RΔ vps -IΔ vps -II) are shown; scale bars represent 40 μm. Assays were repeated with at least two biological replicates.
Figure Legend Snippet: Biofilm structure analysis of a vps -clusters deletion mutant. CSLM images of horizontal ( xy ) and vertical ( xz ) projections (large and side panels, respectively) of biofilm structures formed by rugose wild-type and a mutant strain unable to produce VPS (RΔ vps -IΔ vps -II) are shown; scale bars represent 40 μm. Assays were repeated with at least two biological replicates.

Techniques Used: Mutagenesis

Biofilm formation and VPS production in vps mutants. (a) Biofilm-forming capacities of the rugose wild-type and vps mutants were determined using the crystal violet staining assay on cultures grown at 30 °C for 8 h under static conditions. Results are means of at least five technical replicates and error bars represent standard deviations. (b) VPS production by rugose wild-type and vps mutants was determined by immunoblot analysis using crude VPS extract spotted on a nitrocellulose membrane and probed with an anti-VPS antiserum. (c) Quantification of VPS production in rugose wild-type and vps mutants. Purified VPS was used to quantify VPS production in the strains indicated. Results are means of at least three technical replicates and error bars represent standard deviations. Assays were repeated with two biological replicates.
Figure Legend Snippet: Biofilm formation and VPS production in vps mutants. (a) Biofilm-forming capacities of the rugose wild-type and vps mutants were determined using the crystal violet staining assay on cultures grown at 30 °C for 8 h under static conditions. Results are means of at least five technical replicates and error bars represent standard deviations. (b) VPS production by rugose wild-type and vps mutants was determined by immunoblot analysis using crude VPS extract spotted on a nitrocellulose membrane and probed with an anti-VPS antiserum. (c) Quantification of VPS production in rugose wild-type and vps mutants. Purified VPS was used to quantify VPS production in the strains indicated. Results are means of at least three technical replicates and error bars represent standard deviations. Assays were repeated with two biological replicates.

Techniques Used: Staining, Purification

14) Product Images from "Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †"

Article Title: Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.00466-08

Analysis of the cAMP-CRP contribution to VpsR regulation of rbmC and bap1 expression. (A to C) β-Galactosidase assays of wild-type, Δ crp , Δ vpsT , Δ crp Δ vpsT , Δ vpsR , and Δ crp Δ vpsR strains harboring (A) vpsL-lacZ , (B) rbmC-lacZ , and (C) bap1-lacZ fusion constructs. The data are representative of at least two independent experiments. The error bars indicate standard deviations. (D) Quantitative comparison of biofilm formation by wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. The data are representative of two independent experiments. The error bars indicate standard deviations. (E) Biofilms formed after 8 h of incubation at 30°C in a non-flow-cell system by the wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. Biofilms were stained with SYTO9, and images were acquired by CLSM. The large images are images of the upper surfaces of biofilms, and the images below and to right of the large images are orthogonal views. Bars = 40 μm.
Figure Legend Snippet: Analysis of the cAMP-CRP contribution to VpsR regulation of rbmC and bap1 expression. (A to C) β-Galactosidase assays of wild-type, Δ crp , Δ vpsT , Δ crp Δ vpsT , Δ vpsR , and Δ crp Δ vpsR strains harboring (A) vpsL-lacZ , (B) rbmC-lacZ , and (C) bap1-lacZ fusion constructs. The data are representative of at least two independent experiments. The error bars indicate standard deviations. (D) Quantitative comparison of biofilm formation by wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. The data are representative of two independent experiments. The error bars indicate standard deviations. (E) Biofilms formed after 8 h of incubation at 30°C in a non-flow-cell system by the wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. Biofilms were stained with SYTO9, and images were acquired by CLSM. The large images are images of the upper surfaces of biofilms, and the images below and to right of the large images are orthogonal views. Bars = 40 μm.

Techniques Used: Expressing, Construct, Incubation, Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

Phenotypic characterization of GGDEF deletion mutants and GGDEF crp double-deletion mutants. (A) Pellicle formation, (B) quantitative comparison of biofilm formation, and (C) motility assays for the wild type, for Δ crp and GGDEF single-deletion mutants, and for mutants with GGDEF deletions generated in the Δ crp genetic background. The data are representative of two independent experiments. The error bars indicate standard deviations.
Figure Legend Snippet: Phenotypic characterization of GGDEF deletion mutants and GGDEF crp double-deletion mutants. (A) Pellicle formation, (B) quantitative comparison of biofilm formation, and (C) motility assays for the wild type, for Δ crp and GGDEF single-deletion mutants, and for mutants with GGDEF deletions generated in the Δ crp genetic background. The data are representative of two independent experiments. The error bars indicate standard deviations.

Techniques Used: Generated

Model of cAMP-CRP regulation of biofilm formation in V. cholerae . cAMP-CRP regulates biofilm formation at multiple levels. Expression of vps and rbm genes is negatively regulated by cAMP-CRP through positive regulation of hapR expression and through negative regulation of vpsR and vpsT expression. In turn, VpsR and VpsT positively regulate vps and rbm gene expression, while HapR negatively regulates vps and rbm gene expression. VpsR, VpsT, and HapR regulation of vps and rbm gene expression also involves c-di-GMP signaling, where cdgA expression is negatively regulated by HapR and positively regulated by VpsR and VpsT. An increase in cdgA transcription leads to an increase in the c-di-GMP level, which in turn could interact with an effector protein(s) to positively regulate biofilm formation. We have a very limited understanding of the link between c-di-GMP pools and the signaling that leads to biofilm formation in V. cholerae . In addition, cAMP-CRP may also directly regulate vpsR , cdgA , and rbmC expression and indirectly regulate vpsT expression.
Figure Legend Snippet: Model of cAMP-CRP regulation of biofilm formation in V. cholerae . cAMP-CRP regulates biofilm formation at multiple levels. Expression of vps and rbm genes is negatively regulated by cAMP-CRP through positive regulation of hapR expression and through negative regulation of vpsR and vpsT expression. In turn, VpsR and VpsT positively regulate vps and rbm gene expression, while HapR negatively regulates vps and rbm gene expression. VpsR, VpsT, and HapR regulation of vps and rbm gene expression also involves c-di-GMP signaling, where cdgA expression is negatively regulated by HapR and positively regulated by VpsR and VpsT. An increase in cdgA transcription leads to an increase in the c-di-GMP level, which in turn could interact with an effector protein(s) to positively regulate biofilm formation. We have a very limited understanding of the link between c-di-GMP pools and the signaling that leads to biofilm formation in V. cholerae . In addition, cAMP-CRP may also directly regulate vpsR , cdgA , and rbmC expression and indirectly regulate vpsT expression.

Techniques Used: Expressing

15) Product Images from "Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents"

Article Title: Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

Journal: Molecules

doi: 10.3390/molecules15020780

4-days-old biofilms of Gfp-tagged wild-type grown in FAB medium without (A) or with 25 µM EGCG (B). Biofilms were further treated with 50 µg/ml ciprofloxacin for 24 h (C: biofilm grown in FAB without EGCG; D: biofilm grown in FAB with 25 µM EGCG), after which they were stained with propidium iodide and images were acquired by CLSM. Live cells appear green and dead cells appear red.
Figure Legend Snippet: 4-days-old biofilms of Gfp-tagged wild-type grown in FAB medium without (A) or with 25 µM EGCG (B). Biofilms were further treated with 50 µg/ml ciprofloxacin for 24 h (C: biofilm grown in FAB without EGCG; D: biofilm grown in FAB with 25 µM EGCG), after which they were stained with propidium iodide and images were acquired by CLSM. Live cells appear green and dead cells appear red.

Techniques Used: Staining, Confocal Laser Scanning Microscopy

16) Product Images from "Statistical Analysis of Pseudomonas aeruginosa Biofilm Development: Impact of Mutations in Genes Involved in Twitching Motility, Cell-to-Cell Signaling, and Stationary-Phase Sigma Factor Expression"

Article Title: Statistical Analysis of Pseudomonas aeruginosa Biofilm Development: Impact of Mutations in Genes Involved in Twitching Motility, Cell-to-Cell Signaling, and Stationary-Phase Sigma Factor Expression

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.4.2008-2017.2002

Spatial structures of 98-h-old biofilms of wild-type P. aeruginosa PAO1 (A), P. aeruginosa rpoS (B), P. aeruginosa Δ pilHIJK (C), and P. aeruginosa lasI (D) growing in citrate minimal medium. The bacteria are expressing the GFP. The larger central plots are simulated fluorescent projections in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. The scale bars shown in the central plots are also valid for the right and lower frames.
Figure Legend Snippet: Spatial structures of 98-h-old biofilms of wild-type P. aeruginosa PAO1 (A), P. aeruginosa rpoS (B), P. aeruginosa Δ pilHIJK (C), and P. aeruginosa lasI (D) growing in citrate minimal medium. The bacteria are expressing the GFP. The larger central plots are simulated fluorescent projections in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. The scale bars shown in the central plots are also valid for the right and lower frames.

Techniques Used: Expressing

Roughness versus average thickness of 98-h-old (A) and 146-h-old (B) biofilms of wild-type P. aeruginosa (•), P. aeruginosa rpoS (▿), P. aeruginosa Δ pilHIJK (▪), and P. aeruginosa lasI (⋄). Images were acquired by CLSM in three independent biofilm experiments and quantified by the computer program COMSTAT. In each of the three rounds, 18 image stacks were acquired from two flow channels totaling 54 image stacks for each strain at each time point. Each spot represents a single stack of images. Note that there are only 36 spots for the P. aeruginosa rpoS mutant at the 98-h time point because, inadvertently, no images were acquired for this strain at the 98-h time point in the second round.
Figure Legend Snippet: Roughness versus average thickness of 98-h-old (A) and 146-h-old (B) biofilms of wild-type P. aeruginosa (•), P. aeruginosa rpoS (▿), P. aeruginosa Δ pilHIJK (▪), and P. aeruginosa lasI (⋄). Images were acquired by CLSM in three independent biofilm experiments and quantified by the computer program COMSTAT. In each of the three rounds, 18 image stacks were acquired from two flow channels totaling 54 image stacks for each strain at each time point. Each spot represents a single stack of images. Note that there are only 36 spots for the P. aeruginosa rpoS mutant at the 98-h time point because, inadvertently, no images were acquired for this strain at the 98-h time point in the second round.

Techniques Used: Confocal Laser Scanning Microscopy, Flow Cytometry, Mutagenesis

17) Product Images from "Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms"

Article Title: Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms

Journal: Applied and Environmental Microbiology

doi:

On-line monitoring of establishment and changes in microbial activity in a flow chamber biofilm consisting of SM1639 [ P. putida R1::P rrnB P1- gfp (AAV)]. The fluorescent signals emitted by the cells were visualized with SCLM. The images in panels A to D were recorded at the same flow chamber position 12, 17, 20, and 24.5 h after inoculation, respectively. Each image is presented as a horizontal cross section through a microcolony of cells located at the glass surface in the flow chamber. All images were recorded with the same SCLM setting. Bar, 10 μm.
Figure Legend Snippet: On-line monitoring of establishment and changes in microbial activity in a flow chamber biofilm consisting of SM1639 [ P. putida R1::P rrnB P1- gfp (AAV)]. The fluorescent signals emitted by the cells were visualized with SCLM. The images in panels A to D were recorded at the same flow chamber position 12, 17, 20, and 24.5 h after inoculation, respectively. Each image is presented as a horizontal cross section through a microcolony of cells located at the glass surface in the flow chamber. All images were recorded with the same SCLM setting. Bar, 10 μm.

Techniques Used: On-line Monitoring, Activity Assay, Flow Cytometry

Demonstration of microbial growth activity in flow chamber monoculture biofilms. Comparison of different monitor strains. The panels represent flow chamber biofilms with the following strains: A, B, and C, JM156 ( P. putida R1 [Nal r ]); D, E, and F, SM1639 [ P. putida R1::P rrnB P1- gfp (AAV)]; G, H, and I, SM1699 ( P. putida R1::P rrnB P1- gfp mut3b*). Panels A, D, and G are bright-field images. Panels B, E, and H are SCLM images of the green fluorescent cells presented as simulated fluorescent projections where a long shadow indicates a large, high microcolony. Panels C, F, and I are vertical sections through the biofilm collected at positions indicated by the white triangles. The substratum is located at the left edge of panels C, F, and I. Vertical sections are included to provide information on vertical microcolony size. All images were recorded with the same SCLM settings, i.e., the same laser power and PMT setting. The bar indicates 10 μm, and the scale applies to all of the panels.
Figure Legend Snippet: Demonstration of microbial growth activity in flow chamber monoculture biofilms. Comparison of different monitor strains. The panels represent flow chamber biofilms with the following strains: A, B, and C, JM156 ( P. putida R1 [Nal r ]); D, E, and F, SM1639 [ P. putida R1::P rrnB P1- gfp (AAV)]; G, H, and I, SM1699 ( P. putida R1::P rrnB P1- gfp mut3b*). Panels A, D, and G are bright-field images. Panels B, E, and H are SCLM images of the green fluorescent cells presented as simulated fluorescent projections where a long shadow indicates a large, high microcolony. Panels C, F, and I are vertical sections through the biofilm collected at positions indicated by the white triangles. The substratum is located at the left edge of panels C, F, and I. Vertical sections are included to provide information on vertical microcolony size. All images were recorded with the same SCLM settings, i.e., the same laser power and PMT setting. The bar indicates 10 μm, and the scale applies to all of the panels.

Techniques Used: Activity Assay, Flow Cytometry

Comparison of microbial activity in flow chamber biofilms monitored either as green fluorescence [Gfp(AAV) expression] emitted from strain SM1639 ( P. putida R1:: rrnB P1- gfp (AAV)] or by fluorescent rRNA hybridization of the same biofilm cells. Approximately 20 h after initial colonization of a flow chamber, the biofilm was fixed and embedded. The oligonucleotide probe PP986 labeled with CY3 was used for the hybridizations. Panel A represents Gfp(AAV) expression and panel B represents the hybridization signal in a horizontal SCLM cross section of the glass surface-attached cells in such an embedded biofilm. Bar, 10 μm.
Figure Legend Snippet: Comparison of microbial activity in flow chamber biofilms monitored either as green fluorescence [Gfp(AAV) expression] emitted from strain SM1639 ( P. putida R1:: rrnB P1- gfp (AAV)] or by fluorescent rRNA hybridization of the same biofilm cells. Approximately 20 h after initial colonization of a flow chamber, the biofilm was fixed and embedded. The oligonucleotide probe PP986 labeled with CY3 was used for the hybridizations. Panel A represents Gfp(AAV) expression and panel B represents the hybridization signal in a horizontal SCLM cross section of the glass surface-attached cells in such an embedded biofilm. Bar, 10 μm.

Techniques Used: Activity Assay, Flow Cytometry, Fluorescence, Expressing, Hybridization, Labeling

18) Product Images from "Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa"

Article Title: Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa

Journal: mBio

doi: 10.1128/mBio.00453-15

Biofilm architecture of a mutant producing a high mannuronate molar fraction and M-block. This figure shows biofilm formation and architecture of the PDO300Δ alg8 (pBBR1MCS-5: alg8 ) (frame 1) and PDO300Δ alg44 (pBBR1MCS-5: alg44 ) (frame 3) mutants and nonmucoid mutants (frames 2 and 4). In all frames, A, B, and C show, respectively, top views, side views, and a representative of typical highly structured cell communities for that mutant with biovolume-per-area (µm 3 ⋅ µm −2 ) ratio. Both mutants produce alginates with the highest degree of M-block occurrence but very different degrees of acetylation. The PDO300Δ alg44 (pBBR1MCS-5: alg44 ) (frame 3) mutant, which produces highly acetylated alginate, established very dense and highly developed and larger microcolonies than the PDO300Δ alg8 (pBBR1MCS-5: alg8 ) (frame 1) mutant. One explanation for this significance difference is the presence of an additional copy of Alg44 which senses c-di-GMP, which is a common secondary messenger in the cells governing the physiological condition of cells during colonization. However, nonmucoid mutants did not establish highly structured biofilm and microcolonies.
Figure Legend Snippet: Biofilm architecture of a mutant producing a high mannuronate molar fraction and M-block. This figure shows biofilm formation and architecture of the PDO300Δ alg8 (pBBR1MCS-5: alg8 ) (frame 1) and PDO300Δ alg44 (pBBR1MCS-5: alg44 ) (frame 3) mutants and nonmucoid mutants (frames 2 and 4). In all frames, A, B, and C show, respectively, top views, side views, and a representative of typical highly structured cell communities for that mutant with biovolume-per-area (µm 3 ⋅ µm −2 ) ratio. Both mutants produce alginates with the highest degree of M-block occurrence but very different degrees of acetylation. The PDO300Δ alg44 (pBBR1MCS-5: alg44 ) (frame 3) mutant, which produces highly acetylated alginate, established very dense and highly developed and larger microcolonies than the PDO300Δ alg8 (pBBR1MCS-5: alg8 ) (frame 1) mutant. One explanation for this significance difference is the presence of an additional copy of Alg44 which senses c-di-GMP, which is a common secondary messenger in the cells governing the physiological condition of cells during colonization. However, nonmucoid mutants did not establish highly structured biofilm and microcolonies.

Techniques Used: Mutagenesis, Blocking Assay

Biofilm architecture of mutants producing epimerized and nonepimerized alginates. This figure shows biofilm formation and architecture of the PDO300Δ algG (pBBR1MCS-5: algG ) (frame 1) and PDO300Δ algG (pBBR1MCS-5: algG (D324A)) (frames 3 and 4) mutants, which produce, respectively, epimerized [poly(MG)] and nonepimerized [poly(M)] alginates, and the PDO300Δ algG (pBBR1MCS-5) (frame 2) mutant with no alginate production. In all frames, A, B, and C show, respectively, top views, side views, and a representative of typical highly structured cell communities for that mutant with biovolume-per-area (µm 3 ⋅ µm −2 ) ratio. In frame 3, poly(M) alginate-based biofilm is more highly developed than poly(MG) alginate-based biofilm in frame 1, presenting larger biovolume and biovolume-per-area ratios. Cells of both mutants covered the entire cover slide surface. Frames 4D and E represent the architecture of poly(M) alginate-based microcolonies in which two adjacent structures are connected with horizontal appendages and free-cell void cavities channeled underneath of microcolonies. Frame 4E shows 6 different slices of microcolonies with connected structures at the middle of the figures surrounded by free-cell and matrix areas. Frame 2 represents the homogenous cell community of a nonmucoid mutant without highly structured architecture.
Figure Legend Snippet: Biofilm architecture of mutants producing epimerized and nonepimerized alginates. This figure shows biofilm formation and architecture of the PDO300Δ algG (pBBR1MCS-5: algG ) (frame 1) and PDO300Δ algG (pBBR1MCS-5: algG (D324A)) (frames 3 and 4) mutants, which produce, respectively, epimerized [poly(MG)] and nonepimerized [poly(M)] alginates, and the PDO300Δ algG (pBBR1MCS-5) (frame 2) mutant with no alginate production. In all frames, A, B, and C show, respectively, top views, side views, and a representative of typical highly structured cell communities for that mutant with biovolume-per-area (µm 3 ⋅ µm −2 ) ratio. In frame 3, poly(M) alginate-based biofilm is more highly developed than poly(MG) alginate-based biofilm in frame 1, presenting larger biovolume and biovolume-per-area ratios. Cells of both mutants covered the entire cover slide surface. Frames 4D and E represent the architecture of poly(M) alginate-based microcolonies in which two adjacent structures are connected with horizontal appendages and free-cell void cavities channeled underneath of microcolonies. Frame 4E shows 6 different slices of microcolonies with connected structures at the middle of the figures surrounded by free-cell and matrix areas. Frame 2 represents the homogenous cell community of a nonmucoid mutant without highly structured architecture.

Techniques Used: Mutagenesis

Biofilm architecture of mutants producing acetylated and nonacetylated alginates. This figure shows biofilm formation and architecture of the PDO300Δ algX (pBBR1MCS-5: algX ) (frames 1 and 2) and PDO300Δ algX (pBBR1MCS-5: algX (S269A)) (frame 3) mutants, which produce acetylated and nonacetylated alginates, respectively, and the PDO300Δ algX (pBBR1MCS-5) (frame 4) mutant with no alginate production. In all frames, A, B, and C show top views, side views, and a representative of typical highly structured cell community, respectively. The cell community dimensions are provided as µm 3 ⋅ µm −2 .
Figure Legend Snippet: Biofilm architecture of mutants producing acetylated and nonacetylated alginates. This figure shows biofilm formation and architecture of the PDO300Δ algX (pBBR1MCS-5: algX ) (frames 1 and 2) and PDO300Δ algX (pBBR1MCS-5: algX (S269A)) (frame 3) mutants, which produce acetylated and nonacetylated alginates, respectively, and the PDO300Δ algX (pBBR1MCS-5) (frame 4) mutant with no alginate production. In all frames, A, B, and C show top views, side views, and a representative of typical highly structured cell community, respectively. The cell community dimensions are provided as µm 3 ⋅ µm −2 .

Techniques Used: Mutagenesis

Biofilm architecture of mutant-producing nonepimerized and nonacetylated alginates and the wild type. This figure shows biofilm formation and architecture of the PDO300Δ algX Δ algG (pBBR1MCS-5: algX (S269A): algG (D324A)) (frames 1 and 2) and PDO300(pBBR1MCS-5) (frame 3) mutants. In all frames, A, B, and C show, respectively, top views, side views, and a representative of typical highly structured cell communities for that mutant with biovolume-per-area (µm 3 ⋅ µm −2 ) ratio. The biofilm architecture visualized for the mutant producing nonacetylated poly(M) alginate (frames 1and 2) was remarkably different from that of other applied mutants. Affected by alginate properties, emerging biofilm consists of very narrow but long elevated microcolonies representing longitudinal cell trails or strips indicating stigmergic self-organization and adaptation of cells in weak matrices. Frames 2D to F represent close side and top views of one of the microcolonies and cell trails, and cell-cell interactions in each cell trail are depicted in sketches. Frames 2G to J show micrographs (×40 magnification) of the edge (H and I) and surface (G and J) of mucoid colonies of the PDO300Δ algX Δ algG (pBBR1MCS-5: algX (S269A): algG (D324A)) (G and H) and PDO300(pBBR1MCS-5) (I and J) mutants forming a thin layer on PIA medium after incubation at 37°C for 18 h. Organization of cells of the PDO300Δ algX Δ algG (pBBR1MCS-5: algX (S269A): algG (D324A)) mutant showed a linear filamentous aggregation pattern. Wild-type biofilm architecture is presented in frame 3.
Figure Legend Snippet: Biofilm architecture of mutant-producing nonepimerized and nonacetylated alginates and the wild type. This figure shows biofilm formation and architecture of the PDO300Δ algX Δ algG (pBBR1MCS-5: algX (S269A): algG (D324A)) (frames 1 and 2) and PDO300(pBBR1MCS-5) (frame 3) mutants. In all frames, A, B, and C show, respectively, top views, side views, and a representative of typical highly structured cell communities for that mutant with biovolume-per-area (µm 3 ⋅ µm −2 ) ratio. The biofilm architecture visualized for the mutant producing nonacetylated poly(M) alginate (frames 1and 2) was remarkably different from that of other applied mutants. Affected by alginate properties, emerging biofilm consists of very narrow but long elevated microcolonies representing longitudinal cell trails or strips indicating stigmergic self-organization and adaptation of cells in weak matrices. Frames 2D to F represent close side and top views of one of the microcolonies and cell trails, and cell-cell interactions in each cell trail are depicted in sketches. Frames 2G to J show micrographs (×40 magnification) of the edge (H and I) and surface (G and J) of mucoid colonies of the PDO300Δ algX Δ algG (pBBR1MCS-5: algX (S269A): algG (D324A)) (G and H) and PDO300(pBBR1MCS-5) (I and J) mutants forming a thin layer on PIA medium after incubation at 37°C for 18 h. Organization of cells of the PDO300Δ algX Δ algG (pBBR1MCS-5: algX (S269A): algG (D324A)) mutant showed a linear filamentous aggregation pattern. Wild-type biofilm architecture is presented in frame 3.

Techniques Used: Mutagenesis, Incubation

19) Product Images from "A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials"

Article Title: A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials

Journal: Journal of Antimicrobial Chemotherapy

doi: 10.1093/jac/dkt211

Relationship between CPC concentration and changes in biofilm architecture
Figure Legend Snippet: Relationship between CPC concentration and changes in biofilm architecture

Techniques Used: Concentration Assay

Representative biofilm renderings following each treatment. Green signal indicates viable live cells (Syto 9), red signal indicates damaged/dead cells (propidium iodide). Upper images (A 1 –H 1 ) are of the x–y plane. Middle images (A 2 –H
Figure Legend Snippet: Representative biofilm renderings following each treatment. Green signal indicates viable live cells (Syto 9), red signal indicates damaged/dead cells (propidium iodide). Upper images (A 1 –H 1 ) are of the x–y plane. Middle images (A 2 –H

Techniques Used:

Average percentage signal from biofilms accounted for by ‘live’/viable signal (light grey bars) and ‘dead’/damaged signal (dark grey bars) in relation to total signal captured for both. A sigmoidal relationship was observed
Figure Legend Snippet: Average percentage signal from biofilms accounted for by ‘live’/viable signal (light grey bars) and ‘dead’/damaged signal (dark grey bars) in relation to total signal captured for both. A sigmoidal relationship was observed

Techniques Used:

Epifluorescence images of observed cell shapes and arrangements demonstrating the multi-species composition of the biofilm in the microfluidic system. (a) Chains of cocci. (b) Aggregated rod-shaped cells with outlying diplococci. (c) Fusiforms co-localized
Figure Legend Snippet: Epifluorescence images of observed cell shapes and arrangements demonstrating the multi-species composition of the biofilm in the microfluidic system. (a) Chains of cocci. (b) Aggregated rod-shaped cells with outlying diplococci. (c) Fusiforms co-localized

Techniques Used:

The microfluidic confocal scanning laser microscope biofilm system. (a) Diagram depicting a vertical cross-section of the BioFlux microfluidic system mounted on a Leica SPE CLSM. (b) Annotated photograph showing the channels for two microfluidic channels
Figure Legend Snippet: The microfluidic confocal scanning laser microscope biofilm system. (a) Diagram depicting a vertical cross-section of the BioFlux microfluidic system mounted on a Leica SPE CLSM. (b) Annotated photograph showing the channels for two microfluidic channels

Techniques Used: Microscopy, Confocal Laser Scanning Microscopy

Average percentage abundance of each phylum (a) and genus (b) based on bacterial tag-encoded FLX amplicon pyrosequencing of dental plaque biofilms grown in three randomly harvested BioFlux microfluidic channels.
Figure Legend Snippet: Average percentage abundance of each phylum (a) and genus (b) based on bacterial tag-encoded FLX amplicon pyrosequencing of dental plaque biofilms grown in three randomly harvested BioFlux microfluidic channels.

Techniques Used: Amplification

20) Product Images from "Spatiotemporal pharmacodynamics of meropenem- and tobramycin-treated Pseudomonas aeruginosa biofilms"

Article Title: Spatiotemporal pharmacodynamics of meropenem- and tobramycin-treated Pseudomonas aeruginosa biofilms

Journal: Journal of Antimicrobial Chemotherapy

doi: 10.1093/jac/dkx288

72 h P. aeruginosa PAO1 treated with: (I) three bolus doses of meropenem (2 g) administered every 8 h; (II) three bolus doses of tobramycin (700 mg) administered every 24 h; and (III) meropenem continuous infusion (20 mg/L) with one bolus dose of tobramycin (700 mg). Green cells correspond to live cells and red cells correspond to dead cells. All x / y plots are presented as simulated fluorescence projections. Shown to the right of and below the x / y plots are vertical sections through the biofilm.
Figure Legend Snippet: 72 h P. aeruginosa PAO1 treated with: (I) three bolus doses of meropenem (2 g) administered every 8 h; (II) three bolus doses of tobramycin (700 mg) administered every 24 h; and (III) meropenem continuous infusion (20 mg/L) with one bolus dose of tobramycin (700 mg). Green cells correspond to live cells and red cells correspond to dead cells. All x / y plots are presented as simulated fluorescence projections. Shown to the right of and below the x / y plots are vertical sections through the biofilm.

Techniques Used: Fluorescence

Effect of a single dose of meropenem (top panels) or tobramycin (bottom panels) on a 24 h PAO1 biofilm. For meropenem, CLSM images were acquired prior to antibiotic exposure ( t = 24 h) and 1, 4 and 8 h later. For tobramycin, CLSM images were acquired prior to antibiotic exposure ( t = 24 h) and 4, 6 and 24 h later. Green cells correspond to live cells and red cells correspond to dead cells after staining with propidium iodide. All x / y plots are presented as simulated fluorescence projections. Shown to the right of and below the x / y plots are vertical sections through the biofilm.
Figure Legend Snippet: Effect of a single dose of meropenem (top panels) or tobramycin (bottom panels) on a 24 h PAO1 biofilm. For meropenem, CLSM images were acquired prior to antibiotic exposure ( t = 24 h) and 1, 4 and 8 h later. For tobramycin, CLSM images were acquired prior to antibiotic exposure ( t = 24 h) and 4, 6 and 24 h later. Green cells correspond to live cells and red cells correspond to dead cells after staining with propidium iodide. All x / y plots are presented as simulated fluorescence projections. Shown to the right of and below the x / y plots are vertical sections through the biofilm.

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Fluorescence

PD simulations of effects of meropenem and tobramycin on P. aeruginosa PAO1 biofilms grown in flow chambers. 24 h biofilms were treated with either a single dose of meropenem or tobramycin at t = 24 h. 72 h biofilms were treated with either three doses of meropenem (at t = 72, 80 and 88 h) or tobramycin (at t = 72, 96 and 120 h) or a combination of meropenem and tobramycin (at t = 72 h) MEM, meropenem; TOB, tobramycin.
Figure Legend Snippet: PD simulations of effects of meropenem and tobramycin on P. aeruginosa PAO1 biofilms grown in flow chambers. 24 h biofilms were treated with either a single dose of meropenem or tobramycin at t = 24 h. 72 h biofilms were treated with either three doses of meropenem (at t = 72, 80 and 88 h) or tobramycin (at t = 72, 96 and 120 h) or a combination of meropenem and tobramycin (at t = 72 h) MEM, meropenem; TOB, tobramycin.

Techniques Used: Flow Cytometry

PAO1 biofilm formation in flow chambers in the absence of antibiotic exposure (control). CLSM images were acquired at t = 24, 48, 72, 80, 88, 96, 120 and 144 h. All x / y plots are presented as simulated fluorescence projections. Shown to the right of and below the x / y plots are vertical sections through the biofilm.
Figure Legend Snippet: PAO1 biofilm formation in flow chambers in the absence of antibiotic exposure (control). CLSM images were acquired at t = 24, 48, 72, 80, 88, 96, 120 and 144 h. All x / y plots are presented as simulated fluorescence projections. Shown to the right of and below the x / y plots are vertical sections through the biofilm.

Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy, Fluorescence

21) Product Images from "Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms"

Article Title: Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms

Journal: Nature Communications

doi: 10.1038/ncomms10750

The role of pili and QS in the development of antibiotic-tolerant subpopulations in biofilms. ( a ) Biofilms were cultivated for 72 h using P. aeruginosa PAO1, Δ pilA , Δ pilA /pDA2, Δ lasI Δ rhlI , Δ lasI Δ rhlI +OdDHL and Δ pilA Δ lasR Δ rhlR strains, followed by treatment with medium containing 10 μg ml −1 colistin. No migration of tolerant subpopulation was observed for Δ pilA mutant biofilms, and the majority of the cells in Δ pilA biofilms were killed by colistin. Complementation of Δ pilA with pDA2 restored the antibiotic-tolerant subpopuation development after colistin treatment. The QS-defective Δ lasI Δ rhlI mutant biofilms could only develop small amount of antibiotic-tolerant subpopulation after colistin treatment. Addition of OdDHL partially restored the development of antibiotic-tolerant subpopulation after colistin treatment. The pili and QS-defective Δ pilA Δ lasR Δ rhlR mutant biofilms were unable to develop antibiotic-tolerant subpopulation after colistin treatment. The central images show horizontal optical sections, whereas the flanking images show vertical optical sections. Live cells appear green and dead cells appear red. Scale bars, 50 μm. ( b ) Live/dead ratios of 72-h biofilms formed by P. aeruginosa PAO1, Δ pilA , Δ pilA /pDA2, Δ lasI Δ rhlI , Δ lasI Δ rhlI +OdDHL and Δ pilA Δ lasR Δ rhlR strains after colistin treatment. The mean and s.d. from three experiments is shown. * P
Figure Legend Snippet: The role of pili and QS in the development of antibiotic-tolerant subpopulations in biofilms. ( a ) Biofilms were cultivated for 72 h using P. aeruginosa PAO1, Δ pilA , Δ pilA /pDA2, Δ lasI Δ rhlI , Δ lasI Δ rhlI +OdDHL and Δ pilA Δ lasR Δ rhlR strains, followed by treatment with medium containing 10 μg ml −1 colistin. No migration of tolerant subpopulation was observed for Δ pilA mutant biofilms, and the majority of the cells in Δ pilA biofilms were killed by colistin. Complementation of Δ pilA with pDA2 restored the antibiotic-tolerant subpopuation development after colistin treatment. The QS-defective Δ lasI Δ rhlI mutant biofilms could only develop small amount of antibiotic-tolerant subpopulation after colistin treatment. Addition of OdDHL partially restored the development of antibiotic-tolerant subpopulation after colistin treatment. The pili and QS-defective Δ pilA Δ lasR Δ rhlR mutant biofilms were unable to develop antibiotic-tolerant subpopulation after colistin treatment. The central images show horizontal optical sections, whereas the flanking images show vertical optical sections. Live cells appear green and dead cells appear red. Scale bars, 50 μm. ( b ) Live/dead ratios of 72-h biofilms formed by P. aeruginosa PAO1, Δ pilA , Δ pilA /pDA2, Δ lasI Δ rhlI , Δ lasI Δ rhlI +OdDHL and Δ pilA Δ lasR Δ rhlR strains after colistin treatment. The mean and s.d. from three experiments is shown. * P

Techniques Used: Migration, Mutagenesis

Workflow of pulsed-SILAC proteome analysis of antibiotic-tolerant and -sensitive subpopulations. ( a ) Biofilms were grown in medium containing C12 L -lysine for 72 h and were then treated with medium containing 10 μg ml −1 colistin for 6 h to allow the development of colistin-tolerant cells. Colistin-tolerant subpopulations were then treated with medium containing C13 L -lysine and 10 μg ml −1 colistin to label the tolerant cells with C13. Cells were collected, and pulsed-SILAC proteome analysis was conducted to determine the protein abundance in the colistin-tolerant cells in which the new synthesized proteins are tagged with C13 L -lysine, while the proteome of colistin-susceptible biofilm cells contain the normal C12 L -lysine. Downregulated: lowly expressed proteins in the colistin-tolerant cells ( b ); and upregulated: highly expressed proteins in the colistin-tolerant cells ( c ); proteins in antibiotic-tolerant cells were classified into function groups.
Figure Legend Snippet: Workflow of pulsed-SILAC proteome analysis of antibiotic-tolerant and -sensitive subpopulations. ( a ) Biofilms were grown in medium containing C12 L -lysine for 72 h and were then treated with medium containing 10 μg ml −1 colistin for 6 h to allow the development of colistin-tolerant cells. Colistin-tolerant subpopulations were then treated with medium containing C13 L -lysine and 10 μg ml −1 colistin to label the tolerant cells with C13. Cells were collected, and pulsed-SILAC proteome analysis was conducted to determine the protein abundance in the colistin-tolerant cells in which the new synthesized proteins are tagged with C13 L -lysine, while the proteome of colistin-susceptible biofilm cells contain the normal C12 L -lysine. Downregulated: lowly expressed proteins in the colistin-tolerant cells ( b ); and upregulated: highly expressed proteins in the colistin-tolerant cells ( c ); proteins in antibiotic-tolerant cells were classified into function groups.

Techniques Used: Synthesized

Targeting type IV pili and QS simultaneously leads to eradication of antibiotic-tolerant cells. ( a ) Colistin-tolerant cells formed in PAO1 flow cell biofilms after colistin and erythromycin single treatment and combined treatment. Colistin-tolerant cells were unable to form in PAO1 biofilms after combined erythromycin+colistin treatment. Experiments were performed in triplicate. Live cells appear green and dead cells appear red. Scale bars, 50 μm. ( b ) Live/dead ratios were calculated based on CLSM images. The mean and s.d. from five experiments is shown for in vivo biofilms. * P
Figure Legend Snippet: Targeting type IV pili and QS simultaneously leads to eradication of antibiotic-tolerant cells. ( a ) Colistin-tolerant cells formed in PAO1 flow cell biofilms after colistin and erythromycin single treatment and combined treatment. Colistin-tolerant cells were unable to form in PAO1 biofilms after combined erythromycin+colistin treatment. Experiments were performed in triplicate. Live cells appear green and dead cells appear red. Scale bars, 50 μm. ( b ) Live/dead ratios were calculated based on CLSM images. The mean and s.d. from five experiments is shown for in vivo biofilms. * P

Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy, In Vivo

QS-related products are upregulated in antibiotic-tolerant subpopulations in P. aeruginosa biofilms. ( a ) The 72-h biofilms formed by the P. aeruginosa PAO1 and Δ pilA containing the lasB-gfp translational fusion were treated with medium containing 10 μg ml −1 colistin for 24 h followed by CLSM observation. The lasB-gfp translational fusion was induced to high levels in PAO1 colistin-tolerant cells but was not observed in Δ pilA biofilms. Experiments were performed in triplicate, and a representative image for each condition is shown. Live cells appear green, whereas dead cells appear yellow or red. The central images show horizontal optical sections, whereas the flanking images show vertical optical sections. Scale bars, 50 μm. ( b ) Antibiotic-tolerant cells from PAO1 biofilms secreted more OdDHL than untreated biofilm cells. ( c ) Antibiotic-tolerant cells from PAO1 biofilms produced more elastase than untreated biofilm cells. ( d ) Antibiotic-tolerant cells from PAO1 biofilms produced more pyocyanin than untreated biofilm cells. The mean and s.d. from three experiments is shown. * P
Figure Legend Snippet: QS-related products are upregulated in antibiotic-tolerant subpopulations in P. aeruginosa biofilms. ( a ) The 72-h biofilms formed by the P. aeruginosa PAO1 and Δ pilA containing the lasB-gfp translational fusion were treated with medium containing 10 μg ml −1 colistin for 24 h followed by CLSM observation. The lasB-gfp translational fusion was induced to high levels in PAO1 colistin-tolerant cells but was not observed in Δ pilA biofilms. Experiments were performed in triplicate, and a representative image for each condition is shown. Live cells appear green, whereas dead cells appear yellow or red. The central images show horizontal optical sections, whereas the flanking images show vertical optical sections. Scale bars, 50 μm. ( b ) Antibiotic-tolerant cells from PAO1 biofilms secreted more OdDHL than untreated biofilm cells. ( c ) Antibiotic-tolerant cells from PAO1 biofilms produced more elastase than untreated biofilm cells. ( d ) Antibiotic-tolerant cells from PAO1 biofilms produced more pyocyanin than untreated biofilm cells. The mean and s.d. from three experiments is shown. * P

Techniques Used: Confocal Laser Scanning Microscopy, Produced

Model of antibiotic-tolerant cell formation in P. aeruginosa biofilms. ( a ) Antibiotics such as colistin treatment kill most of the cells in the biofilms, but leave a few antibiotic-tolerant cells at the bottom of the biofilm. ( b ) Antibiotic-tolerant cells expand in numbers and migrate to the top of the biofilm using pilus-mediated motility. ( c ) Assemblies of antibiotic-tolerant cells induce QS that leads to the production of QS-related virulence factors, such as elastase and pyocyanin. A new antibiotic-tolerant biofilm is formed.
Figure Legend Snippet: Model of antibiotic-tolerant cell formation in P. aeruginosa biofilms. ( a ) Antibiotics such as colistin treatment kill most of the cells in the biofilms, but leave a few antibiotic-tolerant cells at the bottom of the biofilm. ( b ) Antibiotic-tolerant cells expand in numbers and migrate to the top of the biofilm using pilus-mediated motility. ( c ) Assemblies of antibiotic-tolerant cells induce QS that leads to the production of QS-related virulence factors, such as elastase and pyocyanin. A new antibiotic-tolerant biofilm is formed.

Techniques Used:

The migration and formation of colistin-tolerant subpopulations in biofilm. ( a ) P. aeruginosa PAO1 wild-type biofilms were treated with minimal medium containing 10 μg ml −1 colistin followed by real-time CLSM observation from 2 to 32 h of treatment. Scale bars, 50 μm. Colistin-tolerant cells in P. aeruginosa PAO1 biofilms migrated onto the dead biofilm and formed a live colistin-tolerant biofilm. Culture medium flow through on top of the biofilms from the top of image. Experiments were performed in triplicate, and a representative image for each condition is shown. Live cells appear green, whereas dead cells appear red or yellow. Videos of the migration and formation of colistin-tolerant biofilm are available in online Supplementary Videos 1 and 2. ( b ) C.f.u. per ml of the PAO1 biofilms after 0, 6, 24 and 48 h of colistin treatment. The means and s.d. from three experiments were shown. ( c ) Movement trajectories and track displacement of live colistin-tolerant cell aggregates moving onto the dead biofilm. Culture medium flow through on top of the biofilms from top of image. Scale bars, 10 μm.
Figure Legend Snippet: The migration and formation of colistin-tolerant subpopulations in biofilm. ( a ) P. aeruginosa PAO1 wild-type biofilms were treated with minimal medium containing 10 μg ml −1 colistin followed by real-time CLSM observation from 2 to 32 h of treatment. Scale bars, 50 μm. Colistin-tolerant cells in P. aeruginosa PAO1 biofilms migrated onto the dead biofilm and formed a live colistin-tolerant biofilm. Culture medium flow through on top of the biofilms from the top of image. Experiments were performed in triplicate, and a representative image for each condition is shown. Live cells appear green, whereas dead cells appear red or yellow. Videos of the migration and formation of colistin-tolerant biofilm are available in online Supplementary Videos 1 and 2. ( b ) C.f.u. per ml of the PAO1 biofilms after 0, 6, 24 and 48 h of colistin treatment. The means and s.d. from three experiments were shown. ( c ) Movement trajectories and track displacement of live colistin-tolerant cell aggregates moving onto the dead biofilm. Culture medium flow through on top of the biofilms from top of image. Scale bars, 10 μm.

Techniques Used: Migration, Confocal Laser Scanning Microscopy, Flow Cytometry

22) Product Images from "The Transcriptional Regulator, CosR, Controls Compatible Solute Biosynthesis and Transport, Motility and Biofilm Formation in Vibrio cholerae"

Article Title: The Transcriptional Regulator, CosR, Controls Compatible Solute Biosynthesis and Transport, Motility and Biofilm Formation in Vibrio cholerae

Journal: Environmental microbiology

doi: 10.1111/j.1462-2920.2012.02805.x

CosR activates biofilm formation in seawater microcosms
Figure Legend Snippet: CosR activates biofilm formation in seawater microcosms

Techniques Used:

CosR activates biofilm formation in artificial seawater
Figure Legend Snippet: CosR activates biofilm formation in artificial seawater

Techniques Used:

23) Product Images from "Discovery and Biological Characterization of the Auromomycin Chromophore as an Inhibitor of Biofilm Formation in Vibrio cholerae"

Article Title: Discovery and Biological Characterization of the Auromomycin Chromophore as an Inhibitor of Biofilm Formation in Vibrio cholerae

Journal: Chembiochem : a European journal of chemical biology

doi: 10.1002/cbic.201300131

Co-dosing concentration curves. IC 50 curves for the inhibition of V. cholerae biofilms by compound 5 alone shown in blue. Shown in red are the IC 50 curves for the inhibition of V. cholerae biofilms treated with varying concentrations of compound 5 plus
Figure Legend Snippet: Co-dosing concentration curves. IC 50 curves for the inhibition of V. cholerae biofilms by compound 5 alone shown in blue. Shown in red are the IC 50 curves for the inhibition of V. cholerae biofilms treated with varying concentrations of compound 5 plus

Techniques Used: Concentration Assay, Inhibition

Examples of natural products reported to possess non-microbicidal biofilm inhibitory activities. Ellagic acid is active against biofilm formation of Staphylococcus aureus with an IC 50 ] Phloretin selectively inhibits Escherichia coli
Figure Legend Snippet: Examples of natural products reported to possess non-microbicidal biofilm inhibitory activities. Ellagic acid is active against biofilm formation of Staphylococcus aureus with an IC 50 ] Phloretin selectively inhibits Escherichia coli

Techniques Used:

Biofilm image analysis. A) Confocal laser scanning microscopic images of V. cholerae at increasing concentrations of compound 5 (Magnification 10×), B) quantitative image analysis conducted using COMSTAT analysis, C) epifluorescence images of
Figure Legend Snippet: Biofilm image analysis. A) Confocal laser scanning microscopic images of V. cholerae at increasing concentrations of compound 5 (Magnification 10×), B) quantitative image analysis conducted using COMSTAT analysis, C) epifluorescence images of

Techniques Used:

Peak library screening results for 1671D. A) Peak library HPLC chromatogram, B) corresponding optical density data and biofilm coverage data acquired for each two-minute time slice of the 1671D prefraction.
Figure Legend Snippet: Peak library screening results for 1671D. A) Peak library HPLC chromatogram, B) corresponding optical density data and biofilm coverage data acquired for each two-minute time slice of the 1671D prefraction.

Techniques Used: Library Screening, High Performance Liquid Chromatography

24) Product Images from "Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect"

Article Title: Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03551-14

Shear force differentiates the anthranilate effect according to developmental stage. (A) Initial attachment of PAO1 cells was observed in the early stage of biofilm development (15 h after inoculation) with indole or anthranilate treatment. The fluorescence
Figure Legend Snippet: Shear force differentiates the anthranilate effect according to developmental stage. (A) Initial attachment of PAO1 cells was observed in the early stage of biofilm development (15 h after inoculation) with indole or anthranilate treatment. The fluorescence

Techniques Used: Fluorescence

Anthranilate deteriorates the biofilm structure. (A) Biofilm of PAO1 cells formed in a flow cell system with treatment of anthranilate or indole. At 5 days after inoculation, biofilms were directly observed with a bright-field microscope (BF) or stained
Figure Legend Snippet: Anthranilate deteriorates the biofilm structure. (A) Biofilm of PAO1 cells formed in a flow cell system with treatment of anthranilate or indole. At 5 days after inoculation, biofilms were directly observed with a bright-field microscope (BF) or stained

Techniques Used: Flow Cytometry, Microscopy, Staining

The effect of anthranilate is QS independent. (A) MW1 harboring pAB1 was used for biofilm formation. The biofilms of GFP-expressing MW1 cells were grown with 0.1 mM anthranilate (AA) or 0.4 mM indole (IN) in a flow cell system for 24 h. Biofilms were
Figure Legend Snippet: The effect of anthranilate is QS independent. (A) MW1 harboring pAB1 was used for biofilm formation. The biofilms of GFP-expressing MW1 cells were grown with 0.1 mM anthranilate (AA) or 0.4 mM indole (IN) in a flow cell system for 24 h. Biofilms were

Techniques Used: Expressing, Flow Cytometry

Anthranilate enhances static biofilm formation. Static biofilm assay was carried out as described in Materials and Methods. Wild-type PAO1 cells were grown in 96-well plates and the measurement was taken at 24 h after inoculation. -, DMSO as a buffer
Figure Legend Snippet: Anthranilate enhances static biofilm formation. Static biofilm assay was carried out as described in Materials and Methods. Wild-type PAO1 cells were grown in 96-well plates and the measurement was taken at 24 h after inoculation. -, DMSO as a buffer

Techniques Used: Biofilm Production Assay

Biofilm-enhancing effect of indole is QS independent. PAO1 (wild type [WT]) and MW1 (QS mutant lacking lasI and rhlI ) cells harboring pAB1 were grown in the drip-flow chamber to form biofilms with indole treatment. WT and MW1 cells in the left column
Figure Legend Snippet: Biofilm-enhancing effect of indole is QS independent. PAO1 (wild type [WT]) and MW1 (QS mutant lacking lasI and rhlI ) cells harboring pAB1 were grown in the drip-flow chamber to form biofilms with indole treatment. WT and MW1 cells in the left column

Techniques Used: Mutagenesis, Flow Cytometry

25) Product Images from "Novel Roles for the AIDA Adhesin from Diarrheagenic Escherichia coli: Cell Aggregation and Biofilm Formation"

Article Title: Novel Roles for the AIDA Adhesin from Diarrheagenic Escherichia coli: Cell Aggregation and Biofilm Formation

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.23.8058-8065.2004

Spatial distribution of biofilm formation for Gfp-labeled E. coli strains OS70 (vector control) (A), OS68 (Ag43 + ), (B), and OS71 (AAH-AIDA + ) (C). Biofilm development was monitored by confocal scanning laser microscopy at 15 h (left panels) and 30 h (right panels) after inoculation. The images are representative horizontal sections collected within each biofilm and vertical sections (to the right and below of each larger panel, representing the yz plane and the xz plane, respectively) at the positions indicated by the white lines.
Figure Legend Snippet: Spatial distribution of biofilm formation for Gfp-labeled E. coli strains OS70 (vector control) (A), OS68 (Ag43 + ), (B), and OS71 (AAH-AIDA + ) (C). Biofilm development was monitored by confocal scanning laser microscopy at 15 h (left panels) and 30 h (right panels) after inoculation. The images are representative horizontal sections collected within each biofilm and vertical sections (to the right and below of each larger panel, representing the yz plane and the xz plane, respectively) at the positions indicated by the white lines.

Techniques Used: Labeling, Plasmid Preparation, Microscopy

26) Product Images from "Overexpression of VpsS, a Hybrid Sensor Kinase, Enhances Biofilm Formation in Vibrio cholerae ▿ ▿ †"

Article Title: Overexpression of VpsS, a Hybrid Sensor Kinase, Enhances Biofilm Formation in Vibrio cholerae ▿ ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.00401-09

VpsS modulates biofilm formation.
Figure Legend Snippet: VpsS modulates biofilm formation.

Techniques Used:

Overexpression of vpsS activates biofilm formation. (A) Quantitative comparison of biofilm formation by wild-type strains harboring the vector or p vpsS . Strains were grown for 8 h in LB medium supplemented with ampicillin in the absence (open bars) or
Figure Legend Snippet: Overexpression of vpsS activates biofilm formation. (A) Quantitative comparison of biofilm formation by wild-type strains harboring the vector or p vpsS . Strains were grown for 8 h in LB medium supplemented with ampicillin in the absence (open bars) or

Techniques Used: Over Expression, Plasmid Preparation

27) Product Images from "Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation"

Article Title: Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17071033

The confocal laser scanning microscope (CLSM) images and relative gene expression of C. albicans in biofilms: ( a ) The CLSM images of C. albicans in multi-species biofilms, C. albicans dyed red, representative hyphae form of C. albicans was marked with white arrows; ( b ) Relative gene expression in C. albicans . Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: The confocal laser scanning microscope (CLSM) images and relative gene expression of C. albicans in biofilms: ( a ) The CLSM images of C. albicans in multi-species biofilms, C. albicans dyed red, representative hyphae form of C. albicans was marked with white arrows; ( b ) Relative gene expression in C. albicans . Values are significantly different when labelled with different letters ( p

Techniques Used: Laser-Scanning Microscopy, Confocal Laser Scanning Microscopy, Expressing

Biofilm structural and metabolic analyses: ( a ) The 3D reconstruction of 72 h multi-species biofilm in different DMADDM containing groups, live microbes dyed green, dead microbes dyed red, adjacent live and dead microbes were presented as yellow when they were merged; ( b ) The biomass of 72 h multi-species biofilm; ( c ) The thickness of 72 h multi-species biofilms in different DMADDM containing groups; ( d ) The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results of multi-species biofilms formed on different DMADDM containing denture base resin. Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: Biofilm structural and metabolic analyses: ( a ) The 3D reconstruction of 72 h multi-species biofilm in different DMADDM containing groups, live microbes dyed green, dead microbes dyed red, adjacent live and dead microbes were presented as yellow when they were merged; ( b ) The biomass of 72 h multi-species biofilm; ( c ) The thickness of 72 h multi-species biofilms in different DMADDM containing groups; ( d ) The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results of multi-species biofilms formed on different DMADDM containing denture base resin. Values are significantly different when labelled with different letters ( p

Techniques Used:

Colony forming unit (CFU) counts of multi-species biofilms: ( a ) The total CFU counts of 72 h multi-species biofilms in different dimethylaminododecyl methacrylate (DMADDM) containing groups; ( b ) The C. albicans CFU counts of 72 h multi-species biofilms in different group. Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: Colony forming unit (CFU) counts of multi-species biofilms: ( a ) The total CFU counts of 72 h multi-species biofilms in different dimethylaminododecyl methacrylate (DMADDM) containing groups; ( b ) The C. albicans CFU counts of 72 h multi-species biofilms in different group. Values are significantly different when labelled with different letters ( p

Techniques Used:

Cell damage based on lactate dehydrogenase (LDH) activity and relative gene expression of TR-146 cell: ( a ) Cell damage based on LDH activity after co-culture with biofilm formed on different DMADDM containing groups; ( b ) Relative gene expression of TR-146 cell after it was co-cultured with biofilms of different groups. Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: Cell damage based on lactate dehydrogenase (LDH) activity and relative gene expression of TR-146 cell: ( a ) Cell damage based on LDH activity after co-culture with biofilm formed on different DMADDM containing groups; ( b ) Relative gene expression of TR-146 cell after it was co-cultured with biofilms of different groups. Values are significantly different when labelled with different letters ( p

Techniques Used: Activity Assay, Expressing, Co-Culture Assay, Cell Culture

28) Product Images from "Role of the LytSR Two-Component Regulatory System in Staphylococcus lugdunensis Biofilm Formation and Pathogenesis"

Article Title: Role of the LytSR Two-Component Regulatory System in Staphylococcus lugdunensis Biofilm Formation and Pathogenesis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.00039

Analysis of S. lugdunensis biofilm by confocal laser scanning microscopy (CLSM). The 24 h mature biofilms of (A) S. lugdunensis WT, (B) Δ lytSR , (C) Δ lytSR (pCU1: lytSR ), and (D) Δ lytSR (pCU1) were visualized after Live/Dead staining under CLSM. Live cells stained with SYTO®9 appear in green, while dead cells stained with propidium iodide are in red. Three-dimensional structural images were reconstructed, and the amount of fluorescence of viable and dead cells was determined using Imaris software. The figures represent one of three independent experiments.
Figure Legend Snippet: Analysis of S. lugdunensis biofilm by confocal laser scanning microscopy (CLSM). The 24 h mature biofilms of (A) S. lugdunensis WT, (B) Δ lytSR , (C) Δ lytSR (pCU1: lytSR ), and (D) Δ lytSR (pCU1) were visualized after Live/Dead staining under CLSM. Live cells stained with SYTO®9 appear in green, while dead cells stained with propidium iodide are in red. Three-dimensional structural images were reconstructed, and the amount of fluorescence of viable and dead cells was determined using Imaris software. The figures represent one of three independent experiments.

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Fluorescence, Software

29) Product Images from "The origin of extracellular DNA in bacterial biofilm infections in vivo"

Article Title: The origin of extracellular DNA in bacterial biofilm infections in vivo

Journal: Pathogens and Disease

doi: 10.1093/femspd/ftaa018

NE antibody staining of the murine implant model. CSLM images of deparaffinized sections of silicone implants from the murine implant model 24 h post-insertion. The sections were stained with primary antibodies specific for NE. The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D) , Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E) , Are only the SYTO9 staining. (C, F) , Are only the antibody staining. The images represent two implants. The letter B indicates biofilm. Images represents staining of 2–4 sections obtained from two biological samples (two implants).
Figure Legend Snippet: NE antibody staining of the murine implant model. CSLM images of deparaffinized sections of silicone implants from the murine implant model 24 h post-insertion. The sections were stained with primary antibodies specific for NE. The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D) , Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E) , Are only the SYTO9 staining. (C, F) , Are only the antibody staining. The images represent two implants. The letter B indicates biofilm. Images represents staining of 2–4 sections obtained from two biological samples (two implants).

Techniques Used: Staining

Immunostaining of CF lung tissue. CSLM images of deparaffinized sections of CF lung tissue. The sections were stained with primary antibodies specific for citrullinated histone H3 (citH3) (A-C) . Histone H3 (D-F) or NE (G-I) . The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D, G), Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E, H), Are only the SYTO9 staining. (C, F, I), Are only the antibody staining. The images represent staining of 2–4 sections from three different CF lungs. B indicates biofilm. J: Co-localization of biofilm with antibodies in CF lungs shown as Manders’ co-localization coefficient. ‘Outside biofilm’ are areas containing PMNs and ‘biofilms’ are ROI defined biofilms. Sample size (n): NE outside biofilms (16), NE biofilms (20), H3 outside biofilms (18), H3 biofilms (17), citH3 outside biofilms (15), citH3 biofilms (18). There was significant difference between H3 outside biofilms and H3 biofilms ( P
Figure Legend Snippet: Immunostaining of CF lung tissue. CSLM images of deparaffinized sections of CF lung tissue. The sections were stained with primary antibodies specific for citrullinated histone H3 (citH3) (A-C) . Histone H3 (D-F) or NE (G-I) . The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D, G), Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E, H), Are only the SYTO9 staining. (C, F, I), Are only the antibody staining. The images represent staining of 2–4 sections from three different CF lungs. B indicates biofilm. J: Co-localization of biofilm with antibodies in CF lungs shown as Manders’ co-localization coefficient. ‘Outside biofilm’ are areas containing PMNs and ‘biofilms’ are ROI defined biofilms. Sample size (n): NE outside biofilms (16), NE biofilms (20), H3 outside biofilms (18), H3 biofilms (17), citH3 outside biofilms (15), citH3 biofilms (18). There was significant difference between H3 outside biofilms and H3 biofilms ( P

Techniques Used: Immunostaining, Staining

H3 antibody staining of the murine implant model. CSLM images of deparaffinized sections of silicone implants from the murine implant model 24 h post-insertion. The sections were stained with primary antibodies specific for histone H3 (H3). The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D) , Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E) , Is only the SYTO9 staining. (C, F) , is only the antibody staining. The images represent two implants. The letter B indicates biofilm. Images represents staining of 2–4 sections obtained from two biological samples (two implants).
Figure Legend Snippet: H3 antibody staining of the murine implant model. CSLM images of deparaffinized sections of silicone implants from the murine implant model 24 h post-insertion. The sections were stained with primary antibodies specific for histone H3 (H3). The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D) , Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E) , Is only the SYTO9 staining. (C, F) , is only the antibody staining. The images represent two implants. The letter B indicates biofilm. Images represents staining of 2–4 sections obtained from two biological samples (two implants).

Techniques Used: Staining

PNA FISH and DAPI staining of CF lung tissue. Deparaffinated CF lung tissue section stained with (A) , PNA FISH to show a P. aeruginosa (red) and (B) , DAPI (blue) to show DNA of PMNs and eDNA. (C) , Shows an overlay of A and B. (D) , A deparaffinated CF lung tissue section stained with the DNA stain propidium iodide (PI) to illustrate both biofilm and eDNA. White arrows point to P. aeruginosa biofilm and black arrows point to eDNA as strings. (Bars: 9 μm).
Figure Legend Snippet: PNA FISH and DAPI staining of CF lung tissue. Deparaffinated CF lung tissue section stained with (A) , PNA FISH to show a P. aeruginosa (red) and (B) , DAPI (blue) to show DNA of PMNs and eDNA. (C) , Shows an overlay of A and B. (D) , A deparaffinated CF lung tissue section stained with the DNA stain propidium iodide (PI) to illustrate both biofilm and eDNA. White arrows point to P. aeruginosa biofilm and black arrows point to eDNA as strings. (Bars: 9 μm).

Techniques Used: Fluorescence In Situ Hybridization, Staining

The in vivo biofilm lack PMN-derived DNA 24 h post-insertion in the murine implant model. PMNs in the murine implant model were labeled with Click-iT®, in which a modified thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), is incorporated into DNA during active DNA synthesis in murine immune cells in vivo , and hence will label only DNA originating from murine PMNs. DNA is labeled ex vivo with Alexa Fluor® 647 (pink) and counterstained with SYTO9 (green) on an implant 24 h post-insertion in the peritoneal cavity. The number of PMNs stained with SYTO9 were estimated to 66 whereas the EdU-labeled (pink) PMNs were estimated to 28. Thereby 42% PMNs in the images were EdU labeled. In the PMN accumulations SYTO9 (green)-stained DNA strings (white arrows) were observed. EdU labeling was observed in PMNs (pink), but was absent from biofilms (double headed arrows), suggesting that PMNs are not a source of eDNA in biofilms. (A, D) , merged images showing both EDU (pink) and SYTO9 (green) staining. (B, E) , Images showing only the SYTO9 staining. (C, F) , images showing only the EDU staining. Red squares in A indicates magnified area in D-F and G-I. Images represents staining obtained from four biological samples (four implants).
Figure Legend Snippet: The in vivo biofilm lack PMN-derived DNA 24 h post-insertion in the murine implant model. PMNs in the murine implant model were labeled with Click-iT®, in which a modified thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), is incorporated into DNA during active DNA synthesis in murine immune cells in vivo , and hence will label only DNA originating from murine PMNs. DNA is labeled ex vivo with Alexa Fluor® 647 (pink) and counterstained with SYTO9 (green) on an implant 24 h post-insertion in the peritoneal cavity. The number of PMNs stained with SYTO9 were estimated to 66 whereas the EdU-labeled (pink) PMNs were estimated to 28. Thereby 42% PMNs in the images were EdU labeled. In the PMN accumulations SYTO9 (green)-stained DNA strings (white arrows) were observed. EdU labeling was observed in PMNs (pink), but was absent from biofilms (double headed arrows), suggesting that PMNs are not a source of eDNA in biofilms. (A, D) , merged images showing both EDU (pink) and SYTO9 (green) staining. (B, E) , Images showing only the SYTO9 staining. (C, F) , images showing only the EDU staining. Red squares in A indicates magnified area in D-F and G-I. Images represents staining obtained from four biological samples (four implants).

Techniques Used: In Vivo, Derivative Assay, Labeling, Modification, DNA Synthesis, Ex Vivo, Staining

The in vivo biofilm lack PMN-derived DNA 48 h post-insertion in the murine implant model. PMNs in the murine implant model were labeled with Click-iT®, in which a modified thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), is incorporated into DNA during active DNA synthesis in murine immune cells in vivo , and hence will label only DNA originating from murine PMNs. DNA is labeled ex vivo with Alexa Fluor® 647 (pink) and counterstained with SYTO9 (green) on an implant 48 h post-insertion in the peritoneal cavity. The number of PMNs stained with SYTO9 were estimated to 36 whereas the EdU labeled (pink) PMNs were estimated to 32. Thereby 89% PMNs in the images were EdU labeled. Labeling was observed in PMNs (pink) but was absent from biofilms (double-headed arrows), suggesting that PMNs are not a source of eDNA in biofilms. (A, D) , Merged images showing both EDU (pink) and SYTO9 (green) staining. (B, E) , Images showing only the SYTO9 staining. (C, F) , images showing only the EDU staining. Red square in A indicates magnified area in D-F. Images represents staining obtained from four biological samples (four implants).
Figure Legend Snippet: The in vivo biofilm lack PMN-derived DNA 48 h post-insertion in the murine implant model. PMNs in the murine implant model were labeled with Click-iT®, in which a modified thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), is incorporated into DNA during active DNA synthesis in murine immune cells in vivo , and hence will label only DNA originating from murine PMNs. DNA is labeled ex vivo with Alexa Fluor® 647 (pink) and counterstained with SYTO9 (green) on an implant 48 h post-insertion in the peritoneal cavity. The number of PMNs stained with SYTO9 were estimated to 36 whereas the EdU labeled (pink) PMNs were estimated to 32. Thereby 89% PMNs in the images were EdU labeled. Labeling was observed in PMNs (pink) but was absent from biofilms (double-headed arrows), suggesting that PMNs are not a source of eDNA in biofilms. (A, D) , Merged images showing both EDU (pink) and SYTO9 (green) staining. (B, E) , Images showing only the SYTO9 staining. (C, F) , images showing only the EDU staining. Red square in A indicates magnified area in D-F. Images represents staining obtained from four biological samples (four implants).

Techniques Used: In Vivo, Derivative Assay, Labeling, Modification, DNA Synthesis, Ex Vivo, Staining

(A) , Pseudomonas aeruginosa is internalized by PMNs in the murine implant model. Top row: TEM images showing intact and active PMNs containing internalized P. aeruginosa (arrows) 6 h post-insertion of a pre-coated silicone implant (Bar: 2 μm). Bottom row: internalized bacteria are magnified (Bar: 500 nm). Images represents sections obtained from two biological samples (two implants). (B–D) , Interaction between PMNs and P. aeruginosa in the murine implant model. Pre-coated silicone implants were inserted into the peritoneal cavity of BALB/c mice. The interaction between PMNs and bacteria was imaged by TEM at 6 h (B), 24 h (C) and 48 h (D) post-insertion. In (B) black arrows points to bacteria and in (C) to a P. aeruginosa biofilm. Arrow heads: PMNs (Bar: 5 μm). Images represents sections obtained from two biological samples (two implants). (E-G) , Matrix material surrounding P. aeruginosa on a silicone implant at 24 h post-insertion. TEM images showing matrix material surrounding P. aeruginosa bacteria at different magnifications. Matrix material (black arrows) can be seen between the bacteria. (E) Bar: 2 μm; (F) Bar: 1 μm; and (G) Bar: 200 nm. Images represents sections obtained from two biological samples (two implants).
Figure Legend Snippet: (A) , Pseudomonas aeruginosa is internalized by PMNs in the murine implant model. Top row: TEM images showing intact and active PMNs containing internalized P. aeruginosa (arrows) 6 h post-insertion of a pre-coated silicone implant (Bar: 2 μm). Bottom row: internalized bacteria are magnified (Bar: 500 nm). Images represents sections obtained from two biological samples (two implants). (B–D) , Interaction between PMNs and P. aeruginosa in the murine implant model. Pre-coated silicone implants were inserted into the peritoneal cavity of BALB/c mice. The interaction between PMNs and bacteria was imaged by TEM at 6 h (B), 24 h (C) and 48 h (D) post-insertion. In (B) black arrows points to bacteria and in (C) to a P. aeruginosa biofilm. Arrow heads: PMNs (Bar: 5 μm). Images represents sections obtained from two biological samples (two implants). (E-G) , Matrix material surrounding P. aeruginosa on a silicone implant at 24 h post-insertion. TEM images showing matrix material surrounding P. aeruginosa bacteria at different magnifications. Matrix material (black arrows) can be seen between the bacteria. (E) Bar: 2 μm; (F) Bar: 1 μm; and (G) Bar: 200 nm. Images represents sections obtained from two biological samples (two implants).

Techniques Used: Transmission Electron Microscopy, Mouse Assay

Hypothesis for formation of an eDNA shield in chronic bacterial infections in vivo . Early during biofilm formation, PMNs are able to phagocytose and destroy single bacteria or very small particles of bacterial cells. As the biofilm develops, bacterial aggregates evade phagocytosis and induce a necrotic cell death of PMNs. In chronic bacterial infections PMNs are continuously recruited to the site of biofilms where they release eDNA via necrosis. This released eDNA does not become incorporated into the biofilm itself. The PMN-derived layer of eDNA, which constitutes a secondary matrix, may provide a passive physical shield for the biofilm against cationic antibiotics such as tobramycin and additional phagocytes.
Figure Legend Snippet: Hypothesis for formation of an eDNA shield in chronic bacterial infections in vivo . Early during biofilm formation, PMNs are able to phagocytose and destroy single bacteria or very small particles of bacterial cells. As the biofilm develops, bacterial aggregates evade phagocytosis and induce a necrotic cell death of PMNs. In chronic bacterial infections PMNs are continuously recruited to the site of biofilms where they release eDNA via necrosis. This released eDNA does not become incorporated into the biofilm itself. The PMN-derived layer of eDNA, which constitutes a secondary matrix, may provide a passive physical shield for the biofilm against cationic antibiotics such as tobramycin and additional phagocytes.

Techniques Used: In Vivo, Derivative Assay

citH3 antibody staining of the murine implant model. CSLM images of deparaffinized sections of silicone implants from the murine implant model 24 h post-insertion. The sections were stained with primary antibodies specific for citrullinated histone H3 (citH3). The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D) , Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E) , Are only the SYTO9 staining. (C, F) , are only the antibody staining. The images represent two implants. The letter B indicates biofilm. Images represents staining of 2–4 sections obtained from two biological samples (two implants).
Figure Legend Snippet: citH3 antibody staining of the murine implant model. CSLM images of deparaffinized sections of silicone implants from the murine implant model 24 h post-insertion. The sections were stained with primary antibodies specific for citrullinated histone H3 (citH3). The secondary antibody was conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. (A, D) , Merged images of both the antibody (pink) and SYTO9 (green) staining. (B, E) , Are only the SYTO9 staining. (C, F) , are only the antibody staining. The images represent two implants. The letter B indicates biofilm. Images represents staining of 2–4 sections obtained from two biological samples (two implants).

Techniques Used: Staining

30) Product Images from "Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype"

Article Title: Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03173-14

IMARIS easy 3D projections from CLSM images of the biofilms formed by the 96 isolates of the ListRA collection showing the predominance of the honeycomb-like morphotype. The biofilms were labeled in green with Syto 9 and in red with propidium iodide.
Figure Legend Snippet: IMARIS easy 3D projections from CLSM images of the biofilms formed by the 96 isolates of the ListRA collection showing the predominance of the honeycomb-like morphotype. The biofilms were labeled in green with Syto 9 and in red with propidium iodide.

Techniques Used: Confocal Laser Scanning Microscopy, Labeling

Correlation between the biofilm structural parameters of the ListRA collection. Shown are the distributions of the mean thickness (A) and roughness (B) as a function of the biovolume and the distribution of roughness as a function of the mean thickness
Figure Legend Snippet: Correlation between the biofilm structural parameters of the ListRA collection. Shown are the distributions of the mean thickness (A) and roughness (B) as a function of the biovolume and the distribution of roughness as a function of the mean thickness

Techniques Used:

Microscopic observations of the biofilms formed by the motile L. monocytogenes 10403S WT strain and its isogenic nonmotile 10403S Δ flaA mutant. (A) Isosurface representation obtained from the confocal image series using the IMARIS software (green
Figure Legend Snippet: Microscopic observations of the biofilms formed by the motile L. monocytogenes 10403S WT strain and its isogenic nonmotile 10403S Δ flaA mutant. (A) Isosurface representation obtained from the confocal image series using the IMARIS software (green

Techniques Used: Mutagenesis, Software

as a representative of nonmotile strains forming flat biofilms. (A and B) IMARIS isosurface representation (A) and section view (B) of the
Figure Legend Snippet: as a representative of nonmotile strains forming flat biofilms. (A and B) IMARIS isosurface representation (A) and section view (B) of the

Techniques Used:

Biovolumes of L. monocytogenes EGD-e biofilms depending on their growth conditions. Biofilms were grown for 48 h at 25°C. The media used are shown on the x axis (TSB10, 10× dilution of TSB; glu 1%, addition of 10 g/liter of glucose; MOPS,
Figure Legend Snippet: Biovolumes of L. monocytogenes EGD-e biofilms depending on their growth conditions. Biofilms were grown for 48 h at 25°C. The media used are shown on the x axis (TSB10, 10× dilution of TSB; glu 1%, addition of 10 g/liter of glucose; MOPS,

Techniques Used:

Strain H25 as a representative of motile strains forming biofilms with a honeycomb morphotype. (A and B) IMARIS isosurface representation (A) and section view (B) of CLSM images from biofilms forming honeycomb-like structures stained in green with Syto
Figure Legend Snippet: Strain H25 as a representative of motile strains forming biofilms with a honeycomb morphotype. (A and B) IMARIS isosurface representation (A) and section view (B) of CLSM images from biofilms forming honeycomb-like structures stained in green with Syto

Techniques Used: Confocal Laser Scanning Microscopy, Staining

31) Product Images from "Modulation of Biofilm Exopolysaccharides by the Streptococcus mutans vicX Gene"

Article Title: Modulation of Biofilm Exopolysaccharides by the Streptococcus mutans vicX Gene

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.01432

Dynamics of the expression of exopolysaccharide-associated genes and phenotypic characteristics of S. mutans . (A) Differences in vicR/K/X expression in interaction with the vicRK transduction system among three types of strains. (B) Differences between gtfB / C/D expressions in exopolysaccharide synthesis of three types of strains. (C) Differences in ftf , gbpB , and dexA expression in exopolysaccharide synthetic catalysis and degradation among three types of strains. S. mutans gene expression was relatively quantified by real-time PCR using gyrA as an internal control and calculated based on the UA159 expression, which was set as 1.0. Results were averaged from 10 independent cultures of different strains (UA159, SmuvicX, SmuvicX+) and experiments were performed in triplicate. The data are presented as mean ± standard deviations. Shapiro–Wilk tests and Bartlett's tests showed that the data were nonparametric. The Kruskal–Wallis test and least significant difference (LSD) multiple comparisons method were used to compare the effects of variables. Asterisks indicate the significant differences among the expressions of genes were considered differentially at a minimal ratio of two-fold changes. (D) Scanning electron microscopy (SEM) observation of the architecture of S. mutans biofilms. Images were taken at 2000×, 5000×, and 20,000× magnifications, respectively. Clusters of bacterial cells surrounded by enriched exopolysaccharide matrix in the biofilm of the UA159 and SmuvicX+ strains (yellow arrows). The SmuvicX strain seemed to be devoid of exopolysaccharide matrix in the biofilms. Representative images are shown from three randomly selected areas from each sample. (E) The means of surface roughness average (Ra) of S. mutans biofilms obtained from atomic force microscopy (AFM) experiments were calculated. The values obtained for SmuvicX (61.11 ± 2.55) were lower than those obtained for the UA159 (75.4 ± 4.23) and SmuvicX+ (75.81 ± 4.1) strains. Results were averaged from 10 independent cultures of different strains (UA159, SmuvicX, SmuvicX+) and experiments were performed in triplicate. The data are presented as mean ± standard errors. Shapiro–Wilk tests and Bartlett's tests showed that the data were parametric. One-way ANOVA were used to detect the significant effects of variables, * P
Figure Legend Snippet: Dynamics of the expression of exopolysaccharide-associated genes and phenotypic characteristics of S. mutans . (A) Differences in vicR/K/X expression in interaction with the vicRK transduction system among three types of strains. (B) Differences between gtfB / C/D expressions in exopolysaccharide synthesis of three types of strains. (C) Differences in ftf , gbpB , and dexA expression in exopolysaccharide synthetic catalysis and degradation among three types of strains. S. mutans gene expression was relatively quantified by real-time PCR using gyrA as an internal control and calculated based on the UA159 expression, which was set as 1.0. Results were averaged from 10 independent cultures of different strains (UA159, SmuvicX, SmuvicX+) and experiments were performed in triplicate. The data are presented as mean ± standard deviations. Shapiro–Wilk tests and Bartlett's tests showed that the data were nonparametric. The Kruskal–Wallis test and least significant difference (LSD) multiple comparisons method were used to compare the effects of variables. Asterisks indicate the significant differences among the expressions of genes were considered differentially at a minimal ratio of two-fold changes. (D) Scanning electron microscopy (SEM) observation of the architecture of S. mutans biofilms. Images were taken at 2000×, 5000×, and 20,000× magnifications, respectively. Clusters of bacterial cells surrounded by enriched exopolysaccharide matrix in the biofilm of the UA159 and SmuvicX+ strains (yellow arrows). The SmuvicX strain seemed to be devoid of exopolysaccharide matrix in the biofilms. Representative images are shown from three randomly selected areas from each sample. (E) The means of surface roughness average (Ra) of S. mutans biofilms obtained from atomic force microscopy (AFM) experiments were calculated. The values obtained for SmuvicX (61.11 ± 2.55) were lower than those obtained for the UA159 (75.4 ± 4.23) and SmuvicX+ (75.81 ± 4.1) strains. Results were averaged from 10 independent cultures of different strains (UA159, SmuvicX, SmuvicX+) and experiments were performed in triplicate. The data are presented as mean ± standard errors. Shapiro–Wilk tests and Bartlett's tests showed that the data were parametric. One-way ANOVA were used to detect the significant effects of variables, * P

Techniques Used: Expressing, Transduction, Real-time Polymerase Chain Reaction, Electron Microscopy, Microscopy

The structural properties of the glucan of S. mutans biofilms were further elucidated using 600 MHz 1 H nuclear magnetic resonance (NMR) spectroscopy . Several sharp well-resolved peaks corresponding to the dextran standard metabolites could be observed in each of the strains. The 1 H NMR spectrum of the water-insoluble glucan (WIG) of the UA159 strain consisted mostly of signals at 4.003, 3.992, 3.985, 3.9301, 3.913, 3.755, 3.727, 3.594, 3.588, 3.577, 3.571, 3.544, and 3.513 ppm (A) ; the 1 H NMR spectrum of the water-soluble glucan (WSG) of the UA159 strain consisted mostly of signals at 3.985, 3.9301, 3.913, 3.772, 3.755, 3.743, 3.727, 3.711, 3.577, 3.528, and 3.513 ppm (B) ; the 1 H NMR spectrum of the WIG of the SmuvicX strain consisted of lesser signals at 3.930, 3.772, 3.743, 3.727, 3.594, and 3.544 ppm (C) ; the 1 H NMR spectrum of the WSG of the SmuvicX strain consisted of signals at 4.003, 3.992, 3.985, 3.930, 3.772, 3.743, 3.727, 3.711, 3.577, 3.571, and 3.544 ppm (D) ; the 1 H NMR spectrum of the WIG of the SmuvicX+ strain consisted of signals at 3.992, 3.930, 3.913, 3.772, 3.755, 3.743, 3.727, 3.711, 3.588, and 3.577 ppm (E) ; and the 1 H NMR spectrum of the WSG of the SmuvicX+ strain consisted of signals at 3.992, 3.913, 3.755, 3.743, 3.727, 3.711, 3.571, and 3.544 ppm (F) .
Figure Legend Snippet: The structural properties of the glucan of S. mutans biofilms were further elucidated using 600 MHz 1 H nuclear magnetic resonance (NMR) spectroscopy . Several sharp well-resolved peaks corresponding to the dextran standard metabolites could be observed in each of the strains. The 1 H NMR spectrum of the water-insoluble glucan (WIG) of the UA159 strain consisted mostly of signals at 4.003, 3.992, 3.985, 3.9301, 3.913, 3.755, 3.727, 3.594, 3.588, 3.577, 3.571, 3.544, and 3.513 ppm (A) ; the 1 H NMR spectrum of the water-soluble glucan (WSG) of the UA159 strain consisted mostly of signals at 3.985, 3.9301, 3.913, 3.772, 3.755, 3.743, 3.727, 3.711, 3.577, 3.528, and 3.513 ppm (B) ; the 1 H NMR spectrum of the WIG of the SmuvicX strain consisted of lesser signals at 3.930, 3.772, 3.743, 3.727, 3.594, and 3.544 ppm (C) ; the 1 H NMR spectrum of the WSG of the SmuvicX strain consisted of signals at 4.003, 3.992, 3.985, 3.930, 3.772, 3.743, 3.727, 3.711, 3.577, 3.571, and 3.544 ppm (D) ; the 1 H NMR spectrum of the WIG of the SmuvicX+ strain consisted of signals at 3.992, 3.930, 3.913, 3.772, 3.755, 3.743, 3.727, 3.711, 3.588, and 3.577 ppm (E) ; and the 1 H NMR spectrum of the WSG of the SmuvicX+ strain consisted of signals at 3.992, 3.913, 3.755, 3.743, 3.727, 3.711, 3.571, and 3.544 ppm (F) .

Techniques Used: Nuclear Magnetic Resonance, Spectroscopy

Laser confocal microscopy of the exopolysaccharide matrix in the biofilm architecture and glucan quantification . (A) Double labeling of the biofilms in the three types of strains. Images were taken at 63× magnification. Green, total bacteria (SYTO 9); red, exopolysaccharide (EPS; Alexa Fluor 647); scale bars, 50 μm. The three-dimensional reconstruction of the biofilms was performed using Imaris 7.0.0. (B) Quantitative data of bacterial and exopolysaccharide (EPS) biomass in the three types of strains. Results were averaged from 10 independent cultures of different strains (UA159, SmuvicX, SmuvicX+) and experiments were performed in triplicate. The data are presented as mean ± standard errors. Shapiro–Wilk tests and Bartlett's tests showed that the data were parametric. One-way ANOVA were used to detect the significant effects of variables, * P
Figure Legend Snippet: Laser confocal microscopy of the exopolysaccharide matrix in the biofilm architecture and glucan quantification . (A) Double labeling of the biofilms in the three types of strains. Images were taken at 63× magnification. Green, total bacteria (SYTO 9); red, exopolysaccharide (EPS; Alexa Fluor 647); scale bars, 50 μm. The three-dimensional reconstruction of the biofilms was performed using Imaris 7.0.0. (B) Quantitative data of bacterial and exopolysaccharide (EPS) biomass in the three types of strains. Results were averaged from 10 independent cultures of different strains (UA159, SmuvicX, SmuvicX+) and experiments were performed in triplicate. The data are presented as mean ± standard errors. Shapiro–Wilk tests and Bartlett's tests showed that the data were parametric. One-way ANOVA were used to detect the significant effects of variables, * P

Techniques Used: Confocal Microscopy, Labeling

32) Product Images from "The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin"

Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.187

NTHi 502 static biofilm biomass and planktonic cell viability in the presence of divalent cations. Biofilms were formed in the presence of twofold increases in concentration of Mg + (A), Ba 2+ (B), Ca 2+ (C), Mn 2+ (D), Zn 2+ (E), and Fe 2+ (F) cations in CDM for 24 h at 37°C, 5% CO 2 . Circles represent biofilm biomass. Squares represent planktonic cell growth. Planktonic cell viability was measured by CFU counts and biofilm biomass quantitated with A595 measurement of crystal violet staining. Graphs show values relative to no added cation control. Dotted line indicates 100% of no added cation control values. In the presence of Fe 2+ , Mn 2+ , and Zn 2+ , biofilm formation and planktonic cell viability generally decreased as the cation concentration increased. Biofilms were increased in the presence of Ba 2+ and Ca 2+ in conjunction with an increase in planktonic cell viability. Mg 2+ was the only cation to result in increasing biofilm formation without an increase in planktonic cell viability. NTHi, nontypeable Hemophilus influenzae ; CDM, chemically defined medium pH 9.
Figure Legend Snippet: NTHi 502 static biofilm biomass and planktonic cell viability in the presence of divalent cations. Biofilms were formed in the presence of twofold increases in concentration of Mg + (A), Ba 2+ (B), Ca 2+ (C), Mn 2+ (D), Zn 2+ (E), and Fe 2+ (F) cations in CDM for 24 h at 37°C, 5% CO 2 . Circles represent biofilm biomass. Squares represent planktonic cell growth. Planktonic cell viability was measured by CFU counts and biofilm biomass quantitated with A595 measurement of crystal violet staining. Graphs show values relative to no added cation control. Dotted line indicates 100% of no added cation control values. In the presence of Fe 2+ , Mn 2+ , and Zn 2+ , biofilm formation and planktonic cell viability generally decreased as the cation concentration increased. Biofilms were increased in the presence of Ba 2+ and Ca 2+ in conjunction with an increase in planktonic cell viability. Mg 2+ was the only cation to result in increasing biofilm formation without an increase in planktonic cell viability. NTHi, nontypeable Hemophilus influenzae ; CDM, chemically defined medium pH 9.

Techniques Used: Concentration Assay, Staining

Effect of EDTA and DNaseI in combination with antibiotics on biofilm cell viability. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with EDTA or DNaseI, alone or in combination with ampicillin or ciprofloxacin. Bacterial viability was assessed with live/dead staining kit and imaged using CLSM. Quantitative analysis of dead cell biomass was performed using COMSTAT of the proridium idodide channel (A). Data presented as mean ± SEM (*** P
Figure Legend Snippet: Effect of EDTA and DNaseI in combination with antibiotics on biofilm cell viability. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with EDTA or DNaseI, alone or in combination with ampicillin or ciprofloxacin. Bacterial viability was assessed with live/dead staining kit and imaged using CLSM. Quantitative analysis of dead cell biomass was performed using COMSTAT of the proridium idodide channel (A). Data presented as mean ± SEM (*** P

Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

EDTA or DNaseI enhance the efficacy of ampicillin or ciprofloxacin treatment of established biofilms. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with 25 mmol/L EDTA alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (upper) or with DNaseI (100 Kunitz U/mL) alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (lower). Biofilm biomass was quantitated from CLSM images with COMSTAT. Data presented as mean ± SEM (**** P
Figure Legend Snippet: EDTA or DNaseI enhance the efficacy of ampicillin or ciprofloxacin treatment of established biofilms. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with 25 mmol/L EDTA alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (upper) or with DNaseI (100 Kunitz U/mL) alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (lower). Biofilm biomass was quantitated from CLSM images with COMSTAT. Data presented as mean ± SEM (**** P

Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy

Cation chelators treat established biofilms. To determine the ability of chelators to treat established biofilms, biofilms were formed using static (A–C) or flow (D–F) models of biofilm development, in media supplemented with 2.5 mmol/L Mg 2+ . Biofilms were then treated for 1 h with 25 mmol/L EDTA (B and E) or received no treatment (A and D). Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. CLSM, confocal laser scanning microscopy, EDTA, ethylenediaminetetra-acetic acid.
Figure Legend Snippet: Cation chelators treat established biofilms. To determine the ability of chelators to treat established biofilms, biofilms were formed using static (A–C) or flow (D–F) models of biofilm development, in media supplemented with 2.5 mmol/L Mg 2+ . Biofilms were then treated for 1 h with 25 mmol/L EDTA (B and E) or received no treatment (A and D). Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. CLSM, confocal laser scanning microscopy, EDTA, ethylenediaminetetra-acetic acid.

Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

Mg 2+ enhances NTHi biofilm formation. Biofilms were formed by NTHi 502 in CDM in the absence (A and D) or presence (B and E) of 2.5 mmol/L Mg 2+ using static (A–C) or flow cell (D–F) models of NTHi biofilm develpment. Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. NTHi, nontypeable Hemophilus influenzae ; CDM, chemically defined medium pH 9; CLSM, confocal laser scanning microscopy.
Figure Legend Snippet: Mg 2+ enhances NTHi biofilm formation. Biofilms were formed by NTHi 502 in CDM in the absence (A and D) or presence (B and E) of 2.5 mmol/L Mg 2+ using static (A–C) or flow cell (D–F) models of NTHi biofilm develpment. Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. NTHi, nontypeable Hemophilus influenzae ; CDM, chemically defined medium pH 9; CLSM, confocal laser scanning microscopy.

Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

33) Product Images from "Difference in virulence and composition of a cariogenic biofilm according to substratum direction"

Article Title: Difference in virulence and composition of a cariogenic biofilm according to substratum direction

Journal: Scientific Reports

doi: 10.1038/s41598-018-24626-2

Effect of substratum direction on EPS. ( A ) EPS biovolume, ( B ) EPS thickness of 46-h-old S. mutans biofilm, and ( C ) representative 3-D images of EPSs (red) in the biofilms: (C-1) downward substratum surfaces, (C-2) vertical substratum surfaces, and (C-3) upward substratum surfaces. Values followed by the same superscript are not significantly different from each other ( p > 0.05).
Figure Legend Snippet: Effect of substratum direction on EPS. ( A ) EPS biovolume, ( B ) EPS thickness of 46-h-old S. mutans biofilm, and ( C ) representative 3-D images of EPSs (red) in the biofilms: (C-1) downward substratum surfaces, (C-2) vertical substratum surfaces, and (C-3) upward substratum surfaces. Values followed by the same superscript are not significantly different from each other ( p > 0.05).

Techniques Used:

Acid production in 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Changes in initial rate of H + production (0–20 min) and total produced concentration of H + (180 min) in 46-h-old S. mutans biofilms, calculated from biofilm pH drop assay data. Values followed by the same superscript are not significantly different from each other ( p > 0.05).
Figure Legend Snippet: Acid production in 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Changes in initial rate of H + production (0–20 min) and total produced concentration of H + (180 min) in 46-h-old S. mutans biofilms, calculated from biofilm pH drop assay data. Values followed by the same superscript are not significantly different from each other ( p > 0.05).

Techniques Used: Produced, Concentration Assay

S. mutans biofilm formation on saliva-coated hydroxyapatite (sHA) discs placed in different directions and the experimental plan.
Figure Legend Snippet: S. mutans biofilm formation on saliva-coated hydroxyapatite (sHA) discs placed in different directions and the experimental plan.

Techniques Used:

Effect of substratum direction on live and dead cells. ( A ) Bacterial biovolume, ( B ) bacterial thickness of live and dead cells in 46-h-old S. mutans biofilms, and ( C ) representative 3-D images of live (green) and dead (red) cells in the biofilms: (C-1) downward substratum surfaces, (C-2) vertical substratum surfaces, and (C-3) upward substratum surfaces. Values followed by the same superscript are not significantly different from each other ( p > 0.05). * p
Figure Legend Snippet: Effect of substratum direction on live and dead cells. ( A ) Bacterial biovolume, ( B ) bacterial thickness of live and dead cells in 46-h-old S. mutans biofilms, and ( C ) representative 3-D images of live (green) and dead (red) cells in the biofilms: (C-1) downward substratum surfaces, (C-2) vertical substratum surfaces, and (C-3) upward substratum surfaces. Values followed by the same superscript are not significantly different from each other ( p > 0.05). * p

Techniques Used:

Biofilm density of 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Values followed by the same superscripts are not significantly different from each other ( p > 0.05).
Figure Legend Snippet: Biofilm density of 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Values followed by the same superscripts are not significantly different from each other ( p > 0.05).

Techniques Used:

Dry weight ( A ), CFUs ( B ), and water-insoluble EPSs ( C ) of 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Values followed by the same superscript are not significantly different from each other ( p > 0.05).
Figure Legend Snippet: Dry weight ( A ), CFUs ( B ), and water-insoluble EPSs ( C ) of 46-h-old S. mutans biofilms formed on sHA discs placed in downward, vertical, and upward directions. Values followed by the same superscript are not significantly different from each other ( p > 0.05).

Techniques Used:

Representative SEM images (×100, ×5000) of 46-h-old S. mutans biofilms formed on sHA discs placed in different directions: ( A ) downward substratum surfaces, ( B ) vertical substratum surfaces, and ( C ) upward substratum surfaces.
Figure Legend Snippet: Representative SEM images (×100, ×5000) of 46-h-old S. mutans biofilms formed on sHA discs placed in different directions: ( A ) downward substratum surfaces, ( B ) vertical substratum surfaces, and ( C ) upward substratum surfaces.

Techniques Used:

34) Product Images from "Positive Regulation of Spoilage Potential and Biofilm Formation in Shewanella baltica OS155 via Quorum Sensing System Composed of DKP and Orphan LuxRs"

Article Title: Positive Regulation of Spoilage Potential and Biofilm Formation in Shewanella baltica OS155 via Quorum Sensing System Composed of DKP and Orphan LuxRs

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00135

Scheme representing the QS system in regulating spoilage potential and biofilm formation in S. baltica OS155. At high cell density, QS signal PP reaches a threshold level and is detected by specific receptor LuxR01 and/or LuxR02 protein, which acts as transcription regulator to alter the expression of torS, speF , and pomA . Among them, TorS functions as a signal-transduction cascade mediator and transphosphorylates TorR upon the presence of TMAO, which finally activate torA expression to reduce TMAO into TMA. speF gene is responsible for the production of putrescine from ornithine. pomA gene promotes the biofilm formation by regulating swimming motility.
Figure Legend Snippet: Scheme representing the QS system in regulating spoilage potential and biofilm formation in S. baltica OS155. At high cell density, QS signal PP reaches a threshold level and is detected by specific receptor LuxR01 and/or LuxR02 protein, which acts as transcription regulator to alter the expression of torS, speF , and pomA . Among them, TorS functions as a signal-transduction cascade mediator and transphosphorylates TorR upon the presence of TMAO, which finally activate torA expression to reduce TMAO into TMA. speF gene is responsible for the production of putrescine from ornithine. pomA gene promotes the biofilm formation by regulating swimming motility.

Techniques Used: Expressing, Transduction

Cyclo-( L -Pro- L -Phe) promotes biofilm formation in a LuxR-type protein-dependent manner. (A) Biofilm formation of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was quantified by microtiter plate assay. (B) Biofilm formation of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was analyzed by CLSM imaging in blend mode view (left panel) and section mode view (right panel). The scale bars represent 50 μm. (C) The mean maximum thickness of biofilm formed in wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was calculated by IMARIS software. Data was presented as the mean ± standard deviation ( n = 3, ∗ p
Figure Legend Snippet: Cyclo-( L -Pro- L -Phe) promotes biofilm formation in a LuxR-type protein-dependent manner. (A) Biofilm formation of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was quantified by microtiter plate assay. (B) Biofilm formation of wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was analyzed by CLSM imaging in blend mode view (left panel) and section mode view (right panel). The scale bars represent 50 μm. (C) The mean maximum thickness of biofilm formed in wild-type strain, SB7301 and SB7302 mutants with DMSO (control) and PP treatment was calculated by IMARIS software. Data was presented as the mean ± standard deviation ( n = 3, ∗ p

Techniques Used: Confocal Laser Scanning Microscopy, Imaging, Software, Standard Deviation

Biofilm formation activity of gene deletion mutants. (A) Biofilm formation of wild-type and each deletion mutant (SB7301, SB7302, SB7303, SB7304, SB7305, and SB7306) strains was quantified by microtiter plate assay. (B) Biofilm formation of wild-type, SB7301 and SB7302 mutant strains was analyzed by CLSM imaging in blend mode view (left panel) and section mode view (right panel). The scale bars represent 50 μm. (C) The mean maximum thickness of biofilms obtained from CLSM imaging. Data was presented as the mean ± standard deviation ( n = 3, ∗ p
Figure Legend Snippet: Biofilm formation activity of gene deletion mutants. (A) Biofilm formation of wild-type and each deletion mutant (SB7301, SB7302, SB7303, SB7304, SB7305, and SB7306) strains was quantified by microtiter plate assay. (B) Biofilm formation of wild-type, SB7301 and SB7302 mutant strains was analyzed by CLSM imaging in blend mode view (left panel) and section mode view (right panel). The scale bars represent 50 μm. (C) The mean maximum thickness of biofilms obtained from CLSM imaging. Data was presented as the mean ± standard deviation ( n = 3, ∗ p

Techniques Used: Activity Assay, Mutagenesis, Confocal Laser Scanning Microscopy, Imaging, Standard Deviation

35) Product Images from "Association of blaOXA-23 and bap with the persistence of Acinetobacter baumannii within a major healthcare system"

Article Title: Association of blaOXA-23 and bap with the persistence of Acinetobacter baumannii within a major healthcare system

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00182

Biofilm quantification by various groupings of the population of 290 clinical A. baumannii isolates collected using active surveillance at the Detroit Medical Center between Jan. 2010-May 2011 . The referent boxplot for each panel is the panel on the far left. A * indicates significant difference in mean 560 nm absorbance from referent boxplot ( p
Figure Legend Snippet: Biofilm quantification by various groupings of the population of 290 clinical A. baumannii isolates collected using active surveillance at the Detroit Medical Center between Jan. 2010-May 2011 . The referent boxplot for each panel is the panel on the far left. A * indicates significant difference in mean 560 nm absorbance from referent boxplot ( p

Techniques Used:

Biofilm quantification process of four A. baumannii isolates using crystal violet staining, measurement from a 96-well plate reader, and visualization using confocal laser scanning microscopy . (A) Shows DMC0256, a representative sample bap alone isolate. (B) Shows DMC0420 with both bla OXA-23 and bap . (C) Shows DMC0551, a representative non- bap isolate. A top view, side view, and angled view of the biofilm from each isolate is shown to illustrate biomass, thickness, and confluence differences between the isolates.
Figure Legend Snippet: Biofilm quantification process of four A. baumannii isolates using crystal violet staining, measurement from a 96-well plate reader, and visualization using confocal laser scanning microscopy . (A) Shows DMC0256, a representative sample bap alone isolate. (B) Shows DMC0420 with both bla OXA-23 and bap . (C) Shows DMC0551, a representative non- bap isolate. A top view, side view, and angled view of the biofilm from each isolate is shown to illustrate biomass, thickness, and confluence differences between the isolates.

Techniques Used: Staining, Confocal Laser Scanning Microscopy

36) Product Images from "The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging"

Article Title: The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016177

Biofilm biovolumes and the initial adhesion levels of the seven B.subtilis strains. (A) The biovolumes (µm 3 ) in ascending order of the 48h-biofilms obtained with the seven B. subtilis strains in microtiter plates from confocal image series using the PHLIP tool. (B) Number of cells adhering per cm 2 after 1h30 of adhesion in the microtiter plate. The error bars indicate the standard error and the statistically significant difference observed with strain 168 ( P
Figure Legend Snippet: Biofilm biovolumes and the initial adhesion levels of the seven B.subtilis strains. (A) The biovolumes (µm 3 ) in ascending order of the 48h-biofilms obtained with the seven B. subtilis strains in microtiter plates from confocal image series using the PHLIP tool. (B) Number of cells adhering per cm 2 after 1h30 of adhesion in the microtiter plate. The error bars indicate the standard error and the statistically significant difference observed with strain 168 ( P

Techniques Used:

Three-dimensional biofilm structures obtained with the seven B.subtilis strains. These images present a representative, aerial, 3D view of the 48h-biofilm structures obtained with the seven B. subtilis strains using a microplate system, obtained from confocal image series using IMARIS software (including the shadow projection on the right). One iso-surface representation of a particular “beanstalk-like” structure is also shown for B. subtilis ND medical .
Figure Legend Snippet: Three-dimensional biofilm structures obtained with the seven B.subtilis strains. These images present a representative, aerial, 3D view of the 48h-biofilm structures obtained with the seven B. subtilis strains using a microplate system, obtained from confocal image series using IMARIS software (including the shadow projection on the right). One iso-surface representation of a particular “beanstalk-like” structure is also shown for B. subtilis ND medical .

Techniques Used: Software

Effect of mutations on the three-dimensional structure of biofilms. Effects of mutations on immersed biofilm structures of the biofilms obtained with different GFP-carrying mutant strains and the corresponding reference wild-type (WT) strain from the confocal image series using the IMARIS software. Images depict an aerial view of 48h-biofilms in the microplate system. The scale bar is 50µm.
Figure Legend Snippet: Effect of mutations on the three-dimensional structure of biofilms. Effects of mutations on immersed biofilm structures of the biofilms obtained with different GFP-carrying mutant strains and the corresponding reference wild-type (WT) strain from the confocal image series using the IMARIS software. Images depict an aerial view of 48h-biofilms in the microplate system. The scale bar is 50µm.

Techniques Used: Mutagenesis, Software

Biofilm biovolumes of the 14 B.subtilis mutant strains. Effects of mutations on the biofilm biovolumes of the 48h-biofilms obtained with the 14 mutants and wild-type strain (GM2812) (GFP-carrying strains). Biovolumes were normalized to wild-type (GM2812) and classified in ascending order. The error bars indicate the standard error and the statistically significant difference in biovolume obtained with the wild-type ( P
Figure Legend Snippet: Biofilm biovolumes of the 14 B.subtilis mutant strains. Effects of mutations on the biofilm biovolumes of the 48h-biofilms obtained with the 14 mutants and wild-type strain (GM2812) (GFP-carrying strains). Biovolumes were normalized to wild-type (GM2812) and classified in ascending order. The error bars indicate the standard error and the statistically significant difference in biovolume obtained with the wild-type ( P

Techniques Used: Mutagenesis

37) Product Images from "Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm"

Article Title: Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027943

Antibiotic tolerance of maturing flow-cell biofilms of P. aeruginosa and aggregates harvested from a static P. aeruginosa culture. Biofilms and aggregates were grown for 24 h to 72-h prior to tobramycin (100 ug/ml) treatment for 24-h. For visualization by CLSM a GFP-tagged PAO1 strain (green) was used and stained with the DNA stain PI (red) for visualizing dead bacteria. Panels A, B and C represent biofilm tolerance on day 1, 2 and 3 respectively andpanels D and E are aggregates on day 1 and 2. Length of size bars: 20 µm.
Figure Legend Snippet: Antibiotic tolerance of maturing flow-cell biofilms of P. aeruginosa and aggregates harvested from a static P. aeruginosa culture. Biofilms and aggregates were grown for 24 h to 72-h prior to tobramycin (100 ug/ml) treatment for 24-h. For visualization by CLSM a GFP-tagged PAO1 strain (green) was used and stained with the DNA stain PI (red) for visualizing dead bacteria. Panels A, B and C represent biofilm tolerance on day 1, 2 and 3 respectively andpanels D and E are aggregates on day 1 and 2. Length of size bars: 20 µm.

Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy, Staining

DNA content in P. aeruginosa cultures. DNA (PI) staining of A - Aggregate harvested from a 48-h old stationary culture stained with PI. B – 3 day old biofilm grown in flow-cell stained with PI. C – GFP-tagged planktonic cells (OD – 0.5) stained with PI. Length of size bar: 15 µm.
Figure Legend Snippet: DNA content in P. aeruginosa cultures. DNA (PI) staining of A - Aggregate harvested from a 48-h old stationary culture stained with PI. B – 3 day old biofilm grown in flow-cell stained with PI. C – GFP-tagged planktonic cells (OD – 0.5) stained with PI. Length of size bar: 15 µm.

Techniques Used: Staining, Flow Cytometry

Tolerance towards PMNs. Flow-cell biofilms (A+B) were grown for 72-h before addition of PMNs and the aggregates (C+D) were grown for 48-h before the addition of PMNs. The images shows that aggregates are not phagocytosed or penetrated by PMNs. For visualization a GFP-tagged PAO1 strain (green) was used and SYTO62 was used to stain the PMNs (red). Arrows point at paralyzed PMNs. Length of size bars: 20 µm.
Figure Legend Snippet: Tolerance towards PMNs. Flow-cell biofilms (A+B) were grown for 72-h before addition of PMNs and the aggregates (C+D) were grown for 48-h before the addition of PMNs. The images shows that aggregates are not phagocytosed or penetrated by PMNs. For visualization a GFP-tagged PAO1 strain (green) was used and SYTO62 was used to stain the PMNs (red). Arrows point at paralyzed PMNs. Length of size bars: 20 µm.

Techniques Used: Flow Cytometry, Staining

Growth rates of flow-cell biofilms and aggregates over time. Growth rates were estimated by quantifying rRNA molecules per rDNA molecules by RT-PCR.
Figure Legend Snippet: Growth rates of flow-cell biofilms and aggregates over time. Growth rates were estimated by quantifying rRNA molecules per rDNA molecules by RT-PCR.

Techniques Used: Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

SEM survey of different P. aeruginosa cultures. SEM of A – Aggregate harvested from a 48-h old stationary culture. B – Details of a 3-day old biofilm grown in flow-cell. C – Details of 48-h old stationary aggregate. D – Planktonic cells (OD 600 = 0.5). Note that the single planktonic cells are difficult to fixate on the specimen during SEM preparation due to the small size (leads to few cells on the specimen).
Figure Legend Snippet: SEM survey of different P. aeruginosa cultures. SEM of A – Aggregate harvested from a 48-h old stationary culture. B – Details of a 3-day old biofilm grown in flow-cell. C – Details of 48-h old stationary aggregate. D – Planktonic cells (OD 600 = 0.5). Note that the single planktonic cells are difficult to fixate on the specimen during SEM preparation due to the small size (leads to few cells on the specimen).

Techniques Used: Flow Cytometry

38) Product Images from "Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists"

Article Title: Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056338

Biofilms of mixed V. cholerae A1552 (open) and Δ hapR strains (hatched) were exposed to predation. The relative fitness of each strain in early (A) and late (B) biofilms of mixed V. cholerae strains was measured by enumeration of CFU (C). Error bars represent standard deviation. Experiments were run in replicates of four and repeated twice.
Figure Legend Snippet: Biofilms of mixed V. cholerae A1552 (open) and Δ hapR strains (hatched) were exposed to predation. The relative fitness of each strain in early (A) and late (B) biofilms of mixed V. cholerae strains was measured by enumeration of CFU (C). Error bars represent standard deviation. Experiments were run in replicates of four and repeated twice.

Techniques Used: Standard Deviation

Biofilm grazing assays of V. cholerae A1552, Δ vpsA and Δ hapR strains. Grazing resistance of early (A) and late (B) biofilms was determined by comparing the biofilm biomass of V. cholerae strains in the absence (open) and in the presence (hatched) of R. nasuta and A. castellanii . Error bars represent standard deviation. The experiment was run in replicates of four and repeated twice.
Figure Legend Snippet: Biofilm grazing assays of V. cholerae A1552, Δ vpsA and Δ hapR strains. Grazing resistance of early (A) and late (B) biofilms was determined by comparing the biofilm biomass of V. cholerae strains in the absence (open) and in the presence (hatched) of R. nasuta and A. castellanii . Error bars represent standard deviation. The experiment was run in replicates of four and repeated twice.

Techniques Used: Standard Deviation

Confocal laser scanning microscopic images of mixed biofilms of V. cholerae A1552 and Δ hapR strains. Three dimensional images of early (A, B) and late (C, D) biofilms in the absence (A, C) and presence (B, D) of predation. (Magnification, ×400; scale bar, 50 µm.) The percentage of each strain in mixed biofilms was calculated using biovolumes determined by IMARIS (E). Experiments were run in replicates of four and repeated twice.
Figure Legend Snippet: Confocal laser scanning microscopic images of mixed biofilms of V. cholerae A1552 and Δ hapR strains. Three dimensional images of early (A, B) and late (C, D) biofilms in the absence (A, C) and presence (B, D) of predation. (Magnification, ×400; scale bar, 50 µm.) The percentage of each strain in mixed biofilms was calculated using biovolumes determined by IMARIS (E). Experiments were run in replicates of four and repeated twice.

Techniques Used:

39) Product Images from "Identification of a calcium-controlled negative regulatory system affecting Vibrio cholerae biofilm formation"

Article Title: Identification of a calcium-controlled negative regulatory system affecting Vibrio cholerae biofilm formation

Journal: Environmental microbiology

doi: 10.1111/j.1462-2920.2009.01923.x

CarS and CarR affect biofilm formation
Figure Legend Snippet: CarS and CarR affect biofilm formation

Techniques Used:

Effect of Ca 2+ on vps gene expression and biofilm formation. (A) Comparison of transcription of vpsA–lacZ , vpsL–lacZ , vpsR–lacZ and vpsT–lacZ genes as determined by β-galactosidase assay in wild-type cells that
Figure Legend Snippet: Effect of Ca 2+ on vps gene expression and biofilm formation. (A) Comparison of transcription of vpsA–lacZ , vpsL–lacZ , vpsR–lacZ and vpsT–lacZ genes as determined by β-galactosidase assay in wild-type cells that

Techniques Used: Expressing

Comparison of biofilms of the wild-type, Δ carS and Δ carR mutant strains.
Figure Legend Snippet: Comparison of biofilms of the wild-type, Δ carS and Δ carR mutant strains.

Techniques Used: Mutagenesis

40) Product Images from "A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes"

Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03224

Deletion of stsR decreases the amount of glucans in S. mutans biofilm. The amounts of (A) water insoluble glucans and (B) water soluble glucans in the biofilms of S. mutans UA159, S. mutans Δ stsR , and complement strain were quantified using the phenol-sulfuric acid method and calculated according to the standard curve. Error bars represent standard deviations based on results from at least three biological replicates. ∗∗ Indicates a significance of P
Figure Legend Snippet: Deletion of stsR decreases the amount of glucans in S. mutans biofilm. The amounts of (A) water insoluble glucans and (B) water soluble glucans in the biofilms of S. mutans UA159, S. mutans Δ stsR , and complement strain were quantified using the phenol-sulfuric acid method and calculated according to the standard curve. Error bars represent standard deviations based on results from at least three biological replicates. ∗∗ Indicates a significance of P

Techniques Used:

Biofilm structure and EPS distribution of S. mutans strains observed by confocal microscopy. (A) Double-labeling of 6 h S. mutans biofilms. Green indicates bacteria (SYTO 9), and red indicates EPS (Alexa Fluor 647). Images were taken at 60× magnification. The three-dimensional reconstruction of the biofilms and the quantification of EPS/bacteria biomass were performed with IMARIS 7.0.0. (B) The ratio of EPS to bacteria at different heights was quantified with COMSTAT. Results are the average of five randomly selected positions of each sample and are presented as mean ± standard deviation. (C–E) Quantification of S. mutans UA159 (C) , S. mutans Δ stsR (D) , and Δ stsR/pDL278-stsR (E) biofilms. EPS biomass was performed with COMSTAT at different heights. Results are the average of five randomly selected positions of each sample and are presented as mean ± standard deviation.
Figure Legend Snippet: Biofilm structure and EPS distribution of S. mutans strains observed by confocal microscopy. (A) Double-labeling of 6 h S. mutans biofilms. Green indicates bacteria (SYTO 9), and red indicates EPS (Alexa Fluor 647). Images were taken at 60× magnification. The three-dimensional reconstruction of the biofilms and the quantification of EPS/bacteria biomass were performed with IMARIS 7.0.0. (B) The ratio of EPS to bacteria at different heights was quantified with COMSTAT. Results are the average of five randomly selected positions of each sample and are presented as mean ± standard deviation. (C–E) Quantification of S. mutans UA159 (C) , S. mutans Δ stsR (D) , and Δ stsR/pDL278-stsR (E) biofilms. EPS biomass was performed with COMSTAT at different heights. Results are the average of five randomly selected positions of each sample and are presented as mean ± standard deviation.

Techniques Used: Confocal Microscopy, Labeling, Standard Deviation

Scanning electron microscopy analysis reveals altered biofilm morphology and decreased biofilm extracellular matrix of S. mutans Δ stsR . Biofilms formed by S. mutans UA159, S. mutans Δ stsR , and complement strain were grown for 6 h and then scanned by scanning electron microscopy (SEM) under (A) 1000× magnification, (B) 5000× magnification, and (C) 20000× magnification.
Figure Legend Snippet: Scanning electron microscopy analysis reveals altered biofilm morphology and decreased biofilm extracellular matrix of S. mutans Δ stsR . Biofilms formed by S. mutans UA159, S. mutans Δ stsR , and complement strain were grown for 6 h and then scanned by scanning electron microscopy (SEM) under (A) 1000× magnification, (B) 5000× magnification, and (C) 20000× magnification.

Techniques Used: Electron Microscopy

Deletion of stsR decreases S. mutans biofilm formation at early stage. S. mutans was cultured in BM supplemented with 1% sucrose for 6, 12, 24, and 48 h. The biofilm biomass was determined by CV staining method. Data from three biological replicates were averaged, and the statistical significance between the stsR mutant, wild-type, and complement strain was determined by Student’s t -test. Error bars represent standard deviations based on results from at least three biological replicates. ∗∗ Indicates a significance of P
Figure Legend Snippet: Deletion of stsR decreases S. mutans biofilm formation at early stage. S. mutans was cultured in BM supplemented with 1% sucrose for 6, 12, 24, and 48 h. The biofilm biomass was determined by CV staining method. Data from three biological replicates were averaged, and the statistical significance between the stsR mutant, wild-type, and complement strain was determined by Student’s t -test. Error bars represent standard deviations based on results from at least three biological replicates. ∗∗ Indicates a significance of P

Techniques Used: Cell Culture, Staining, Mutagenesis

Related Articles

Quantitation Assay:

Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
Article Snippet: .. Biofilm volumetric quantitation were performed using the biovolume determination tool of the IMARIS software package (Bitplane AG, Zürich, Switzerland). ..

Confocal Laser Scanning Microscopy:

Article Title: The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans
Article Snippet: .. A three-dimensional reconstruction of the biofilms from CLSM was analyzed using Imaris 7.0.0 software (Bitplane, Zurich, Switzerland). ..

Generated:

Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function
Article Snippet: .. Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.). .. Images were further processed for display by using the PhotoShop software (Adobe, Mountain View, Calif.).

Article Title: The TibA Adhesin/Invasin from Enterotoxigenic Escherichia coli Is Self Recognizing and Induces Bacterial Aggregation and Biofilm Formation
Article Snippet: .. Vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.). .. Images were further processed for display by using Photoshop software (Adobe, Mountain View, Calif.).

Fluorescence:

Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function
Article Snippet: .. Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.). .. Images were further processed for display by using the PhotoShop software (Adobe, Mountain View, Calif.).

Software:

Article Title: The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans
Article Snippet: .. A three-dimensional reconstruction of the biofilms from CLSM was analyzed using Imaris 7.0.0 software (Bitplane, Zurich, Switzerland). ..

Article Title: Identification and Characterization of OscR, a Transcriptional Regulator Involved in Osmolarity Adaptation in Vibrio cholerae ▿ ▿ †
Article Snippet: .. Three-dimensional images of the biofilms were reconstructed using Imaris software (Bitplane) and quantified using COMSTAT ( ). ..

Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
Article Snippet: .. Biofilm volumetric quantitation were performed using the biovolume determination tool of the IMARIS software package (Bitplane AG, Zürich, Switzerland). ..

Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function
Article Snippet: .. Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.). .. Images were further processed for display by using the PhotoShop software (Adobe, Mountain View, Calif.).

Article Title: A new transformation method with nanographene oxides of antisense carryingyycG RNA improved antibacterial properties on methicillin-resistantStaphylococcus aureus biofilm
Article Snippet: .. A three-dimensional reconstruction of the biofilm was created and analyzed by Imaris 7.0.0 software (Imaris 7.0.0, Bitplane, Zurich, Switzerland). ..

Article Title: The TibA Adhesin/Invasin from Enterotoxigenic Escherichia coli Is Self Recognizing and Induces Bacterial Aggregation and Biofilm Formation
Article Snippet: .. Vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.). .. Images were further processed for display by using Photoshop software (Adobe, Mountain View, Calif.).

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    Bitplane biofilm maximal
    Representative 3D reconstructions of biofilms. Biofilms of wild type TM7x associated XH001, XH001Δ lsrB , and XH001Δ luxS monoculture as well as co-culture when they were associated with TM7x were grown (biological triplicate) as described in the Section “Materials and Methods.” Images were obtained by CLSM and reconstructed using Bitplane: Imaris-Microscopy Image Analysis Software (Bitplane). Reconstructed images led to a view of the same biofilms revealing a significant increase in overall <t>biofilm</t> thickness (height) in the TM7x-associated XH001 relative to XH001. (A,C) and (B,D) represent sagittal (xz) and horizontal (xy) projected images, respectively. The relative fluorescence intensity of the pseudo-colored images is reflected by the scale located in the lower right corner. All scale bars are 10 μm in length.
    Biofilm Maximal, supplied by Bitplane, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bitplane three dimensional biofilm structures reconstructions
    Meningococcal <t>biofilm</t> formation of non piliated strains onto epithelial cells. (A) Quantification of the biomass covering cells infected with pilE mutants (Z5463 gfp Δ pilE and Z5463 gfp Δ pilE ΔMDA). Mutants were grown onto FaDu epithelial cells for 18 hours under constant flow. At least three independent experiments were performed. The results are normalized as a percentage of the mean biomass of Z5463 gfp Δ pilE , which was set to 100%. Error bars indicate the standard errors of the mean (SEM). * p
    Three Dimensional Biofilm Structures Reconstructions, supplied by Bitplane, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Representative 3D reconstructions of biofilms. Biofilms of wild type TM7x associated XH001, XH001Δ lsrB , and XH001Δ luxS monoculture as well as co-culture when they were associated with TM7x were grown (biological triplicate) as described in the Section “Materials and Methods.” Images were obtained by CLSM and reconstructed using Bitplane: Imaris-Microscopy Image Analysis Software (Bitplane). Reconstructed images led to a view of the same biofilms revealing a significant increase in overall biofilm thickness (height) in the TM7x-associated XH001 relative to XH001. (A,C) and (B,D) represent sagittal (xz) and horizontal (xy) projected images, respectively. The relative fluorescence intensity of the pseudo-colored images is reflected by the scale located in the lower right corner. All scale bars are 10 μm in length.

    Journal: Frontiers in Microbiology

    Article Title: Quorum Sensing Modulates the Epibiotic-Parasitic Relationship Between Actinomyces odontolyticus and Its Saccharibacteria epibiont, a Nanosynbacter lyticus Strain, TM7x

    doi: 10.3389/fmicb.2018.02049

    Figure Lengend Snippet: Representative 3D reconstructions of biofilms. Biofilms of wild type TM7x associated XH001, XH001Δ lsrB , and XH001Δ luxS monoculture as well as co-culture when they were associated with TM7x were grown (biological triplicate) as described in the Section “Materials and Methods.” Images were obtained by CLSM and reconstructed using Bitplane: Imaris-Microscopy Image Analysis Software (Bitplane). Reconstructed images led to a view of the same biofilms revealing a significant increase in overall biofilm thickness (height) in the TM7x-associated XH001 relative to XH001. (A,C) and (B,D) represent sagittal (xz) and horizontal (xy) projected images, respectively. The relative fluorescence intensity of the pseudo-colored images is reflected by the scale located in the lower right corner. All scale bars are 10 μm in length.

    Article Snippet: Quantification of Biofilm Maximal Thickness (Height), Biovolume, Biofilm Roughness Correlation (Variance), and Biofilm Continuity Ratio Imaris (Bitplane, Belfast, United Kingdom) biofilm analysis XTension software was used to obtain maximal biovolume thickness (height), biovolume, biofilm roughness correlation (variance), and the biofilm continuity ratio.

    Techniques: Co-Culture Assay, Confocal Laser Scanning Microscopy, Microscopy, Software, Fluorescence

    Quantification of total maximal biofilm thickness (height), biovolume, biofilm roughness correlation and biofilm continuity ratio. Histograms represents (A) Maximum Biofilm Thickness (Height), (B) Biovolume, (C) Biofilm Roughness Correlation, and (D) Biofilm Continuity Ratio, comparing wild type XH001, XH001Δ lsrB , and XH001Δ luxS monoculture as well as co-culture when they were associated with TM7x. The images analyzed were acquired with the same settings as indicated in this figure and in section “Materials and Methods”. Three independent cultures for each group were selected with 3 representative images between each culture. Each bar represents the average of two independent cultures performed in triplicate ( error bars , SD). Significance is indicated when p

    Journal: Frontiers in Microbiology

    Article Title: Quorum Sensing Modulates the Epibiotic-Parasitic Relationship Between Actinomyces odontolyticus and Its Saccharibacteria epibiont, a Nanosynbacter lyticus Strain, TM7x

    doi: 10.3389/fmicb.2018.02049

    Figure Lengend Snippet: Quantification of total maximal biofilm thickness (height), biovolume, biofilm roughness correlation and biofilm continuity ratio. Histograms represents (A) Maximum Biofilm Thickness (Height), (B) Biovolume, (C) Biofilm Roughness Correlation, and (D) Biofilm Continuity Ratio, comparing wild type XH001, XH001Δ lsrB , and XH001Δ luxS monoculture as well as co-culture when they were associated with TM7x. The images analyzed were acquired with the same settings as indicated in this figure and in section “Materials and Methods”. Three independent cultures for each group were selected with 3 representative images between each culture. Each bar represents the average of two independent cultures performed in triplicate ( error bars , SD). Significance is indicated when p

    Article Snippet: Quantification of Biofilm Maximal Thickness (Height), Biovolume, Biofilm Roughness Correlation (Variance), and Biofilm Continuity Ratio Imaris (Bitplane, Belfast, United Kingdom) biofilm analysis XTension software was used to obtain maximal biovolume thickness (height), biovolume, biofilm roughness correlation (variance), and the biofilm continuity ratio.

    Techniques: Co-Culture Assay

    Characteristics of P. aeruginosa wild-type (filled circles) and mucA 22 mutant (open circles) biofilms. The biomass content, biofilm thickness, roughness coefficient, and substratum coverage were calculated by the COMSTAT image analysis software from scanning confocal image data. The values are averages of six image stacks acquired in four separate experiments. The biomass content is calculated as the biomass volume (μm 3 ) per substratum surface area (μm 2 ). The roughness coefficient describes the variation in biofilm thickness and is a measure of biofilm heterogeneity. The substratum coverage is the fraction of the substratum area covered by biomass. The times at which the biofilms were assayed were 1, 2, 4, 6, and 8 days (as indicated on the x axis).

    Journal: Journal of Bacteriology

    Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function

    doi: 10.1128/JB.183.18.5395-5401.2001

    Figure Lengend Snippet: Characteristics of P. aeruginosa wild-type (filled circles) and mucA 22 mutant (open circles) biofilms. The biomass content, biofilm thickness, roughness coefficient, and substratum coverage were calculated by the COMSTAT image analysis software from scanning confocal image data. The values are averages of six image stacks acquired in four separate experiments. The biomass content is calculated as the biomass volume (μm 3 ) per substratum surface area (μm 2 ). The roughness coefficient describes the variation in biofilm thickness and is a measure of biofilm heterogeneity. The substratum coverage is the fraction of the substratum area covered by biomass. The times at which the biofilms were assayed were 1, 2, 4, 6, and 8 days (as indicated on the x axis).

    Article Snippet: Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.).

    Techniques: Mutagenesis, Software

    Biofilm formation assay comparing the total biofilm biomasses of PAO1, PDO300, and algDmucA strains after 10 h. Biofilms were prepared and stained as described in Materials and Methods. Both PDO300 and the algDmucA double mutant harbored less biomass in their biofilms than did PAO1. Each value was the average of 32 individual replicates. Avg, average; St. dev., standard deviation.

    Journal: Journal of Bacteriology

    Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function

    doi: 10.1128/JB.183.18.5395-5401.2001

    Figure Lengend Snippet: Biofilm formation assay comparing the total biofilm biomasses of PAO1, PDO300, and algDmucA strains after 10 h. Biofilms were prepared and stained as described in Materials and Methods. Both PDO300 and the algDmucA double mutant harbored less biomass in their biofilms than did PAO1. Each value was the average of 32 individual replicates. Avg, average; St. dev., standard deviation.

    Article Snippet: Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.).

    Techniques: Tube Formation Assay, Staining, Mutagenesis, Standard Deviation

    Epifluorescence and scanning confocal photomicrographs of the surface-attached communities formed by P. aeruginosa wild type and an isogenic alginate-overproducing mutant. The strains are engineered to contain a gfp expression cassette inserted into the chromosome. The biofilms are grown in once flow-through continuous-culture reaction vessels. (Top) Epifluorescence photomicrographs of the wild type (PAO1) and the mucA 22 mutant (PDO300); images were acquired 8 h postinoculation of the biofilm reactor. (Middle) Epifluorescence photomicrographs acquired 24 h postinoculation. (Bottom) Scanning confocal photomicrographs of 5-day-old wild-type and mucA 22 mutant biofilms. The larger central plots are simulated fluorescent projections, in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. Bar, 20 μm.

    Journal: Journal of Bacteriology

    Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function

    doi: 10.1128/JB.183.18.5395-5401.2001

    Figure Lengend Snippet: Epifluorescence and scanning confocal photomicrographs of the surface-attached communities formed by P. aeruginosa wild type and an isogenic alginate-overproducing mutant. The strains are engineered to contain a gfp expression cassette inserted into the chromosome. The biofilms are grown in once flow-through continuous-culture reaction vessels. (Top) Epifluorescence photomicrographs of the wild type (PAO1) and the mucA 22 mutant (PDO300); images were acquired 8 h postinoculation of the biofilm reactor. (Middle) Epifluorescence photomicrographs acquired 24 h postinoculation. (Bottom) Scanning confocal photomicrographs of 5-day-old wild-type and mucA 22 mutant biofilms. The larger central plots are simulated fluorescent projections, in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. Bar, 20 μm.

    Article Snippet: Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.).

    Techniques: Mutagenesis, Expressing, Flow Cytometry

    Increased tobramycin resistance of a P. aeruginosa PDO300 biofilm. (A) Viable biomass content of gfp-expressing P. aeruginosa wild-type and PDO300 mutant biofilms after 24 h of exposure to 2.0 μg of tobramycin per ml (open bars) and nontreated controls (filled bars). gfp fluorescence is a marker of cell viability and allows quantification of the viable biomass by COMSTAT image analysis of SCLM image data. (B) Visualization of live (green fluorescence) and dead (red fluorescence) cells by LIVE/DEAD Bac Light bacterial viability staining kit. The treated biofilms were exposed to tobramycin as described above. Viability was measured by GFP fluorescence (A) and by SYTO 9 viability staining (B).

    Journal: Journal of Bacteriology

    Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function

    doi: 10.1128/JB.183.18.5395-5401.2001

    Figure Lengend Snippet: Increased tobramycin resistance of a P. aeruginosa PDO300 biofilm. (A) Viable biomass content of gfp-expressing P. aeruginosa wild-type and PDO300 mutant biofilms after 24 h of exposure to 2.0 μg of tobramycin per ml (open bars) and nontreated controls (filled bars). gfp fluorescence is a marker of cell viability and allows quantification of the viable biomass by COMSTAT image analysis of SCLM image data. (B) Visualization of live (green fluorescence) and dead (red fluorescence) cells by LIVE/DEAD Bac Light bacterial viability staining kit. The treated biofilms were exposed to tobramycin as described above. Viability was measured by GFP fluorescence (A) and by SYTO 9 viability staining (B).

    Article Snippet: Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.).

    Techniques: Expressing, Mutagenesis, Fluorescence, Marker, BAC Assay, Staining

    Assay of tobramycin sensitivity in a rotating-disk biofilm reactor system. ○, P. aeruginosa wild-type biofilm; ●, PDO300 mutant biofilm. The values represent averages of three separate experiments. Biofilms were treated for 5 h. The planktonic MIC of tobramycin for both these strains is 1 μg/ml.

    Journal: Journal of Bacteriology

    Article Title: Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function

    doi: 10.1128/JB.183.18.5395-5401.2001

    Figure Lengend Snippet: Assay of tobramycin sensitivity in a rotating-disk biofilm reactor system. ○, P. aeruginosa wild-type biofilm; ●, PDO300 mutant biofilm. The values represent averages of three separate experiments. Biofilms were treated for 5 h. The planktonic MIC of tobramycin for both these strains is 1 μg/ml.

    Article Snippet: Simulated fluorescence projections and vertical cross sections through the biofilms were generated by using the IMARIS software package (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo2 workstation (Silicon Graphics, Mountain View, Calif.).

    Techniques: Mutagenesis

    BdcA disperses biofilms in static biofilm tests with 96-well plates. (A) Strains as in the Figure 2 caption. Black bars indicate mature biofilm formation (2 h after IPTG induction for each strain), and the gray bars show biofilm formation after dispersal (19 h after IPTG induction for P. aeruginosa/ pMMB206-BdcA and 21.5 h after IPTG induction for P. fluorescens/ pMMB206-BdcA and R. meliloti/ pMMB206-BdcA). For all three strains, 0.1 mM IPTG was added after 24 h incubation to induce bdcA expression. The P. aeruginosa biofilm was formed at 37°C in LB with 250 μg/mL chloramphenicol, the P. fluorescens biofilm was formed at 30°C in LB with 250 μg/mL chloramphenicol, and the R. meliloti biofilm was formed at 30°C in LB with 50 μg/mL chloramphenicol. pMMB206 indicates cells with this plasmid, and BdcA indicates cells containing pMMB206-BdcA. (B) For the dual-species biofilms, R. meliloti (30°C) and P. aeruginosa (37°C) biofilms were formed in LB for 24 h then E. coli DH5α / pMMB206-BdcA/pRK2013 was added. The mixed biofilm was developed for 4 h, then IPTG was added for 26 h to produce BdcA. For the triple species biofilm, the R. meliloti and P. aeruginosa biofilm was formed in LB for 24 h at 30°C, then E. coli DH5α / pMMB206-BdcA/pRK2013 was added. The mixed biofilm was developed for 11 h, then IPTG was added for another 24 h to produce BdcA. Normalized biofilm is indicated by dividing biofilm formation by cell turbidity. pMMB206 (black bars) indicates cells with this plasmid, and BdcA (gray bars) indicates cells containing pMMB206-BdcA. ** indicates statistical significant difference as determined by a Student's t-test ( p

    Journal: BMC Research Notes

    Article Title: Escherichia coli BdcA controls biofilm dispersal in Pseudomonas aeruginosa and Rhizobium meliloti

    doi: 10.1186/1756-0500-4-447

    Figure Lengend Snippet: BdcA disperses biofilms in static biofilm tests with 96-well plates. (A) Strains as in the Figure 2 caption. Black bars indicate mature biofilm formation (2 h after IPTG induction for each strain), and the gray bars show biofilm formation after dispersal (19 h after IPTG induction for P. aeruginosa/ pMMB206-BdcA and 21.5 h after IPTG induction for P. fluorescens/ pMMB206-BdcA and R. meliloti/ pMMB206-BdcA). For all three strains, 0.1 mM IPTG was added after 24 h incubation to induce bdcA expression. The P. aeruginosa biofilm was formed at 37°C in LB with 250 μg/mL chloramphenicol, the P. fluorescens biofilm was formed at 30°C in LB with 250 μg/mL chloramphenicol, and the R. meliloti biofilm was formed at 30°C in LB with 50 μg/mL chloramphenicol. pMMB206 indicates cells with this plasmid, and BdcA indicates cells containing pMMB206-BdcA. (B) For the dual-species biofilms, R. meliloti (30°C) and P. aeruginosa (37°C) biofilms were formed in LB for 24 h then E. coli DH5α / pMMB206-BdcA/pRK2013 was added. The mixed biofilm was developed for 4 h, then IPTG was added for 26 h to produce BdcA. For the triple species biofilm, the R. meliloti and P. aeruginosa biofilm was formed in LB for 24 h at 30°C, then E. coli DH5α / pMMB206-BdcA/pRK2013 was added. The mixed biofilm was developed for 11 h, then IPTG was added for another 24 h to produce BdcA. Normalized biofilm is indicated by dividing biofilm formation by cell turbidity. pMMB206 (black bars) indicates cells with this plasmid, and BdcA (gray bars) indicates cells containing pMMB206-BdcA. ** indicates statistical significant difference as determined by a Student's t-test ( p

    Article Snippet: Biofilm images from nine random positions were visualized with IMARIS confocal software (Bitplane, Zurich, Switzerland) and analyzed by COMSTAT confocal software as previously described [ ].

    Techniques: Incubation, Expressing, Plasmid Preparation

    BdcA disperses P. aeruginosa and R. meliloti biofilms in flow cell experiments . Representative IMARIS images of P. aeruginosa /pMMB206-BdcA/pP25-gfp and P. aeruginosa /pMMB206/pP25-gfp flow cell biofilm formation after 72 h and 120 h of incubation with LB medium at 37°C (A) . IMARIS images of R. meliloti /pMMB206-BdcA/pP25-gfp and R. meliloti /pMMB206/pP25-gfp flow cell biofilm formation after 67 h and 115 h of incubation with LB medium at 30°C (B) . After forming biofilms in LB for 24 h, IPTG (0.1 mM) was added to induce BdcA production from pMMB206-BdcA. Each strain has pP25-gfp for producing GFP to visualize the biofilms, and carbenicillin (50 μg/mL) was added to retain pP25-gfp. Chloramphenicol (250 μg/mL for P. aeruginosa and 50 μg/mL for R. meliloti ) was used to retain the pMMB206-based plasmids. Scale bars represent 10 μm.

    Journal: BMC Research Notes

    Article Title: Escherichia coli BdcA controls biofilm dispersal in Pseudomonas aeruginosa and Rhizobium meliloti

    doi: 10.1186/1756-0500-4-447

    Figure Lengend Snippet: BdcA disperses P. aeruginosa and R. meliloti biofilms in flow cell experiments . Representative IMARIS images of P. aeruginosa /pMMB206-BdcA/pP25-gfp and P. aeruginosa /pMMB206/pP25-gfp flow cell biofilm formation after 72 h and 120 h of incubation with LB medium at 37°C (A) . IMARIS images of R. meliloti /pMMB206-BdcA/pP25-gfp and R. meliloti /pMMB206/pP25-gfp flow cell biofilm formation after 67 h and 115 h of incubation with LB medium at 30°C (B) . After forming biofilms in LB for 24 h, IPTG (0.1 mM) was added to induce BdcA production from pMMB206-BdcA. Each strain has pP25-gfp for producing GFP to visualize the biofilms, and carbenicillin (50 μg/mL) was added to retain pP25-gfp. Chloramphenicol (250 μg/mL for P. aeruginosa and 50 μg/mL for R. meliloti ) was used to retain the pMMB206-based plasmids. Scale bars represent 10 μm.

    Article Snippet: Biofilm images from nine random positions were visualized with IMARIS confocal software (Bitplane, Zurich, Switzerland) and analyzed by COMSTAT confocal software as previously described [ ].

    Techniques: Flow Cytometry, Incubation

    Meningococcal biofilm formation of non piliated strains onto epithelial cells. (A) Quantification of the biomass covering cells infected with pilE mutants (Z5463 gfp Δ pilE and Z5463 gfp Δ pilE ΔMDA). Mutants were grown onto FaDu epithelial cells for 18 hours under constant flow. At least three independent experiments were performed. The results are normalized as a percentage of the mean biomass of Z5463 gfp Δ pilE , which was set to 100%. Error bars indicate the standard errors of the mean (SEM). * p

    Journal: PLoS Pathogens

    Article Title: A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation

    doi: 10.1371/journal.ppat.1006495

    Figure Lengend Snippet: Meningococcal biofilm formation of non piliated strains onto epithelial cells. (A) Quantification of the biomass covering cells infected with pilE mutants (Z5463 gfp Δ pilE and Z5463 gfp Δ pilE ΔMDA). Mutants were grown onto FaDu epithelial cells for 18 hours under constant flow. At least three independent experiments were performed. The results are normalized as a percentage of the mean biomass of Z5463 gfp Δ pilE , which was set to 100%. Error bars indicate the standard errors of the mean (SEM). * p

    Article Snippet: Three-dimensional biofilm structures reconstructions were generated using the IMARIS software package (Bitplane AG).

    Techniques: Infection, Flow Cytometry