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: mBio

Article Title: Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models

doi: 10.1128/mbio.00184-24

Figure Lengend Snippet: Species composition of biofilms grown on hydroxyapatite discs with feast/famine cycles. (A) Schematic of media changes for the feast-and-famine dental biofilm model. (B) Final counts of CFUs for each microbial species at the end of the 5-day experiment. CFUs for biofilms grown in the presence of 0.2 mM NaF are normalized relative to biofilms grown in the presence of 0.2 mM NaCl. Unnormalized counts from the fluoride-treated and control biofilms are shown in Fig. S3. (C) S. mutans CFUs recovered from biofilms of the indicated composition after each treatment in the feast and famine cycle. Statistical significance is shown relative to all-WT fluoride-treated biofilms (open circles). (D). C. albicans CFUs recovered from biofilms of the indicated composition after each treatment in the feast and famine cycle. Statistical significance is shown relative to all-WT biofilms and fluoride-treated biofilms (open circles). (E) S. gordonii CFUs recovered from biofilms of the indicated composition after each treatment in the feast-and-famine cycle. Statistical significance is shown relative to all-WT biofilms and fluoride-treated biofilms (open circles). For all panels, datapoints represent the mean and SEM of three independent experiments ( n = 3). Significance was calculated using a two-way analysis of variance (ANOVA) followed by Fisher’s LSD test. Statistical significance is shown relative to parallel experiments in the presence of 0.2 mm NaCl and is represented as *p < 0.05, **p < 0.01, and *** p < 0.001.

Article Snippet: Hydroxyapatite (HA) discs, each 0.5-inch diameter and 0.08-inch width (Clarkson Chromatography Products Inc, USA) were affixed in a vertical position to the lid of a 24-well microtiter plate using double-sided tape and sterilized using ethylene oxide as described ( ).

Techniques: Control

Journal: mBio

Article Title: Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models

doi: 10.1128/mbio.00184-24

Figure Lengend Snippet: Properties of biofilms grown on hydroxyapatite discs with feast/famine cycles. (A) Media pH was measured after each cycle of feast and famine. (B) Calcium concentration in media was measured after each cycle of feast and famine. For panels A and B, for each biofilm composition, control experiments were performed in the presence of NaCl (black points/lines in panels A and B). These show close correspondence with overlapping points. For the fluoride-treated conditions, statistical significance is shown relative to the chloride-treated biofilms and is represented as * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) Final biofilm mass at the end of the 5-day experiment. (D). Fluoride content of biofilm at the end of the 5-day experiment. For all panels, datapoints represent the mean and SEM of three independent experiments ( n = 3). Significance was calculated using a two-way analysis of variance (ANOVA) followed by Fisher’s LSD test.

Article Snippet: Hydroxyapatite (HA) discs, each 0.5-inch diameter and 0.08-inch width (Clarkson Chromatography Products Inc, USA) were affixed in a vertical position to the lid of a 24-well microtiter plate using double-sided tape and sterilized using ethylene oxide as described ( ).

Techniques: Concentration Assay, Control