Journal: Nutrients

Article Title: Antimicrobial Activity of EPA and DHA against Oral Pathogenic Bacteria Using an In Vitro Multi-Species Subgingival Biofilm Model

doi: 10.3390/nu12092812

Figure Lengend Snippet: Maximum projection of images obtained by confocal laser scanning microscopy (CLSM) of the 72 h biofilms, where the growth of these biofilms was observed on the surfaces of the hydroxyapatite discs after 60 s of exposure: ( A ) to the negative control (phosphate buffer saline); ( B ) to the ethanol solution; ( C ) to the docosahexaenoic acid (DHA) extracts (100 μM concentration) and ( D ) to 0.2% chlorhexidine. Specimens were stained with the LIVE/DEAD ® BacLightTM Bacterial Viability Kit solution, containing SYTO 9 and Propidium Iodide nucleic acid stains. Cells with a compromised membrane that are considered to be dead or dying were stain red (PI), whereas cells with an intact membrane were stain green (SYTO9).

Article Snippet: Sterile calcium hydroxyapatite (HA) discs, of 7 mm of diameter and 1.8 mm (standard deviation, SD = 0.2) of thickness (Clarkson Chromatography Products, Williamsport, PA, USA), were coated with treated saliva for 4 h at 37 °C in sterile plastic tubes, and then placed in the wells of a 24-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany).

Techniques: Confocal Laser Scanning Microscopy, Negative Control, Saline, Concentration Assay, Staining, Membrane

Journal: Nutrients

Article Title: Antimicrobial Activity of EPA and DHA against Oral Pathogenic Bacteria Using an In Vitro Multi-Species Subgingival Biofilm Model

doi: 10.3390/nu12092812

Figure Lengend Snippet: Scanning electron microscopy (SEM) of biofilms with an evolution of 72 h in hydroxyapatite (HA) discs treated with the negative control: phosphate buffer saline (PBS) ( A ), with docosahexaenoic acid (DHA) at 100 µM ( B ), with EtOH ( C ) or with the positive control: 0.2% chlorhexidine (CHX)( D ). A dense bacterial population could be observed on the HA discs treated with PBS ( A ), forming discontinuous layers of bacteria bonded to the discs. Meanwhile, on the biofilms of the discs treated with DHA ( B ), a lower density of cells distributed across the surface of the disc could be seen, and some of these exhibited structural damages. Likewise, on the discs treated with EtOH ( C ) or CHX ( D ), a reduction in the bacterial density present on the surface of the disc could also be observe, although it was lower than that on the discs treated with DHA ( B ). Chains of Aggregatibacter and/or Streptococcus (blue arrow) and fusiform bacilli of the F. nucleatum genus (yellow arrow) could be identified. Magnification ( A – D ): 1500×. The samples were dried by critical points and coated with gold by sputtering.

Article Snippet: Sterile calcium hydroxyapatite (HA) discs, of 7 mm of diameter and 1.8 mm (standard deviation, SD = 0.2) of thickness (Clarkson Chromatography Products, Williamsport, PA, USA), were coated with treated saliva for 4 h at 37 °C in sterile plastic tubes, and then placed in the wells of a 24-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany).

Techniques: Electron Microscopy, Negative Control, Saline, Positive Control, Bacteria

Journal: Nutrients

Article Title: Antimicrobial Activity of EPA and DHA against Oral Pathogenic Bacteria Using an In Vitro Multi-Species Subgingival Biofilm Model

doi: 10.3390/nu12092812

Figure Lengend Snippet: Maximum projection of images obtained by confocal laser scanning microscopy (CLSM) of the 72 h biofilms, where the growth of these biofilms was observed on the surfaces of the hydroxyapatite discs, stained with LIVE/DEAD ® BacLightTM Bacterial Viability Kit, after 60 s of exposure: ( A ) to the negative control (phosphate buffer saline); ( B ) to the ethanol solution; ( C ) to the docosahexaenoic acid (EPA) extracts (100 μM concentration) and ( D ) to 0.2% chlorhexidine. Specimens were stained with the LIVE/DEAD ® BacLightTM Bacterial Viability Kit solution, containing SYTO 9 and Propidium Iodide nucleic acid stains. Cells with a compromised membrane that are considered to be dead or dying were stained red (PI), whereas cells with an intact membrane were stained green (SYTO9).

Article Snippet: Sterile calcium hydroxyapatite (HA) discs, of 7 mm of diameter and 1.8 mm (standard deviation, SD = 0.2) of thickness (Clarkson Chromatography Products, Williamsport, PA, USA), were coated with treated saliva for 4 h at 37 °C in sterile plastic tubes, and then placed in the wells of a 24-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany).

Techniques: Confocal Laser Scanning Microscopy, Staining, Negative Control, Saline, Concentration Assay, Membrane

Journal: Nutrients

Article Title: Antimicrobial Activity of EPA and DHA against Oral Pathogenic Bacteria Using an In Vitro Multi-Species Subgingival Biofilm Model

doi: 10.3390/nu12092812

Figure Lengend Snippet: Scanning electron microscopy (SEM) of biofilms with an evolution of 72 h in hydroxyapatite (HA) discs treated with the negative control: phosphate buffer saline (PBS) ( A ), with docosahexaenoic acid (EPA) at 100 µM ( B ), with ethanol ( C ) or with the positive control: 0.2% chlorhexidine (CHX)( D ). A dense bacterial population could be observed on the HA discs treated with PBS ( A ), forming discontinuous layers of bacteria bonded to the discs. Meanwhile, on the biofilms of the discs treated with EPA ( B ), a lower density of cells distributed across the surface of the disc could be seen, and some of these exhibited structural damages. Likewise, on the discs treated with EtOH ( C ) or CHX ( D ), a reduction could also be observed in the bacterial density present on the surface of the disc, although this reduction was slighter than that on the discs treated with EPA ( B ). Chains of Aggregatibacter and/or Streptococcus (blue arrow) and fusiform bacilli of the F. nucleatum genus (yellow arrow) could be identified. Magnification ( A – D ): 1500×. The samples were dried by critical points and coated with gold by sputtering.

Article Snippet: Sterile calcium hydroxyapatite (HA) discs, of 7 mm of diameter and 1.8 mm (standard deviation, SD = 0.2) of thickness (Clarkson Chromatography Products, Williamsport, PA, USA), were coated with treated saliva for 4 h at 37 °C in sterile plastic tubes, and then placed in the wells of a 24-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany).

Techniques: Electron Microscopy, Negative Control, Saline, Positive Control, Bacteria

Journal: Antibiotics

Article Title: Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

doi: 10.3390/antibiotics3040509

Figure Lengend Snippet: Sub-MIC concentrations of anti-biofilm peptide 1018 triggered biofilm cell death in the hydroxyapatite biofilm model. (A and B) Biofilms were stained using SYTO-9 to stain live cells green and propidium iodide (PI) to stain dead cells red. Merged images showing bacteria stained with both SYTO-9 and PI are also shown, and a yellow or red merged color was observed for dead cells. Samples were then examined using confocal laser scanning microscopy (CLSM). Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz, and xz dimensions). Three-dimensional reconstructions of biofilms are shown on the far right panel (3D image). The scale bar represents 100 µm in length. ( A ) P. aeruginosa PA14 untreated and treated with 10 µg/mL of peptide; ( B ) B. cenocepacia (IIIa) 4813 untreated and treated with peptide. Two independent experiments were performed for each condition; ( C ) SEM images showing the effect of peptide treatment on biofilm cells. P. aeruginosa and B. cenocepacia biofilms were developed on hydroxyapatite (HA) discs for 3 days and treated three times (every 24 h) with 10 µg/mL of peptide 1018. Biofilms were observed using SEM at a magnification of 20,000× operating at 5 kV. Untreated biofilms grown using the HA biofilm model were typically 30–40 μm thick, while biofilm thickness was reduced to 10–20 μm in peptide-treated samples.

Article Snippet: Sterile hydroxyapatite (HA) disks (0.38-inch diameter by 0.06-inch thickness; Clarkson Chromatography Products, Williamsport, PA, USA) were used as a biofilm substrate.

Techniques: Staining, Bacteria, Confocal Laser Scanning Microscopy

Journal: Antibiotics

Article Title: Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

doi: 10.3390/antibiotics3040509

Figure Lengend Snippet: Proportion of dead biofilm cells in hydroxyapatite plates after treatment with sub-MIC levels of peptide 1018. Biofilms were grown in HA discs for 3 days and treated with peptide 1018 every 24 h. After scanning confocal microscopy the volume ratio of red fluorescence (due to the dead cell stain propidium iodide to green (all bacteria stain Syto-9) plus red fluorescence indicated the proportion of killed cells. Three independent assays were performed. Different superscript letters indicate statistically significant differences between groups ( p < 0.05).

Article Snippet: Sterile hydroxyapatite (HA) disks (0.38-inch diameter by 0.06-inch thickness; Clarkson Chromatography Products, Williamsport, PA, USA) were used as a biofilm substrate.

Techniques: Confocal Microscopy, Fluorescence, Staining, Bacteria