Real-time detection of development and thickness of Pseudomonas aeruginosa biofilm
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Objective To conduct real-time observation of living biofilm formation and determine the thickness of the Pseudomonas aeruginosa biofilm.Methods After green fluorescent protein(GFP)expression plasmid was transfected to Pseudomonas aeruginosa,the horizontal section scanning of living biofilm was done in real-time with confocal laser scanning microscope(CLSM).The thickness of living biofilm was determined according to Z axial distance.Results The biofilm formation of Pseudomonas aeruginosa was observed in three stages,which were the adherence phase from 0 to 6 hours,the assembly phase from 6 to 24 hours,and the maturation phase from 24 to 72 hours.The thickness of the biofilm at 6,24,48,and 72 hr were 6.1±2.8,29.2±2.3,61.4±1.4,and 61.8±1.1μm,respectively.Statistical analysis showed that the thickness of the biofilm at 6 hr was less than that of at 24 hr(P0.01)and the thickness of the biofilm at 24 hr was less than that of at 48 hr(P0.01).There was no difference in the thickness of the biofilm between the time of 48 hr and 72 hr(P0.05).Conclusion CLSM can be used to observe the biofilm formation of Pseudomonas aeruginosa in real-time and to determine its thickness.Keywords:
Laser Microscopy
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In order to better study the biofilm formation process, image structure analyzer software was used to quantitatively analyze the biofilm formation process of Pseudomonas aeruginosa under different concentrations of glucose and allicin. Glucose solution (5.6, 7.0, 11.1, 16.7, 20 and 30 mmol/L) was added to the culture medium of non repetitive Pseudomonas aeruginosa and 6 groups were formed. Minimum inhibitory concentration of allicin to Pseudomonas aeruginosa PAO1 was determined by Tryptic Soy Broth micro dilution method. Single colony was inoculated into Tryptic Soy Broth according to garlic concentration (10; 128 μg/mL). Concomitantly, 3 groups were divided into 1, 3 and 7 d study. The groups of experimental materials were placed in confocal laser scanning microscope for observation and the obtained pictures were imported into the image structure analyzer software, through which the quantitative analysis of the biofilm structure was performed. As main findings we found that as glucose concentration increases, the thickness of Pseudomonas aeruginosa biofilm gradually increased. After glucose (30 mmol/L) treatment for 3 d, areal porosity decreased from 0.93±0.10 to 0.62±0.02 and the biofilm became thicker; textural entropy changed from 6.67±0.99 to 7.88±0.21, meaning an increase in biofilm heterogeneity and thickness; the average diffusion distance decreased from 2.44±0.28 to 1.72±0.36, the biofilm became thicker and the average diffusion distance between colonies also decreased. Under the intervention of allicin 10 μg/mL, the thickness of biomembrane was significantly reduced, the structure of biomembrane was sparse and the numbers of dead and living bacteria were significantly reduced. Under the action of allicin 128 μg/mL, the thickness of biomembrane was further reduced and the dead bacteria were clustered. After the action of allicin 128 μg/mL for 7 d, areal porosity increased from 0.68±0.10 to 0.92±0.02, the biomembrane became sparser; textural entropy increased from 6.67±0.93 changed to 5.52±0.548, the membrane heterogeneity decreased. Image structure analyzer software showed to be suitable for quantitative analysis of biofilm formation process. It is concluded that with the increase of glucose concentration, the maturation speed of Pseudomonas aeruginosa biofilm is accelerated, and the thickness of biofilm is gradually increased; allicin can destroy the self structure of Pseudomonas aeruginosa biofilm.
Tryptic soy broth
Dilution
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Objective To establish the model of Staphylococcus epidermidis biofilm in vitro,observe and quantitatively analyze the dynamic procedure of the biofilm formation.Method Biofilm model of biofilm-producing strain Staphylococcus epidermidis RP62A was formed on plate.The metabolic activity of the bacteria inside biofilm was measured by tetrazolium salt(XTT) reduction assay.The parameters of BF structure were analyzed through pictures from Confocal Laser Scanning Microscopy(CLSM) with Image Structure Analyer(ISA) software.The structure of the biofilm was detected by scanning electron microscope(SEM).Result The data of A450 by XTT reduction assay at different time intervals(12 h,24 h,48 h) were 2.392±0.481,3.410±0.177 and 3.917±0.274,respectively.There were significant difference between any two groups(P0.05).The data from ISA software showed that the areal porosity(AP) were the 0.84±0.08, 0.68±0.01 and 0.59±0.13,respectively;the average diffusion distance(ADD) were 1.34±0.24,1.49±0.09 and 1.89±0.39,respectively;There were significant difference between any two groups(P0.05).The textural entropy(TE) were 4.71±0.82,8.69±0.68 and 8.94±0.28,respectively.There were significant difference between the 12h group and the other two groups(P0.05).Conclusion The formation of Staphylococcus epidermidis BF is a dynamic procedure.Mature biofilm was formed at the 24th hour.The structure became more complex at the 48th hour.XTT reduction assay,CLSM with ISA and SEM are the ideal methods to observe and quantitatively analyze the biofilm model in vitro.
Formazan
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Objective To investigate the sequential development of spatial structure of biofilm with Image Structure Analyzer (ISA) software so as to found a basis for further research on biological behavior of biofilm. Methods In vitro biofilm model of Pseudomonas Aeruginosa (P. aeruginosa) PAO1 tagged with SYTO9/PI was established on glass slice, and its biofilm development was monitored at different time points (6 h, 1, 3, and 6 d). The fluorescence images stack of different layers in biofilm model were obtained by confocal laser scanning microscopy (CLSM) based on fluorophores from PAO1. Quantitative parameters describing biofilm spatial structure were acquired after the image information was calculated by ISA software. Results ①The PAO1 biofilm process was investigated successfully by CLSM after genetically tagged with GFP. ②With the development of biofilm, the ratio of died bacteria was increased gradually in each layer and most of them were distributed in the core of microcolony. ③The quantitative data from ISA software showed that the thickness of biofilm was increased significantly during biofilm growth; meanwhile the areal porosity (AP) was decreased significantly, the average diffusion distance (ADD) indicated an increased tendency; and the textural entropy (TE) increased significantly. Conclusion The ISA software combined with SYTO9/PI tagging provide useful information of bacterial biofilm spatial structure in PAO1 biofilm process. Quantifying bacterial biofilm structure permits correlating biofilm development with biofilm performance.
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The growth characteristics of Vibrio parahaemolyticus biofilms on glass surfaces, the effects of environmental factors during biofilm formation, and biofilm detachment under ultrasound treatment were investigated. The morphology of V. parahaemolyticus biofilms on the glass surface was observed by crystal violet staining, biofilm biomass was measured using an enzyme-linked immunosorbent assay(ELISA) plate reader at 595 nm, and the extent of biofilm detachment was measured by counting colony-forming units(CFU) using the plate count method. V. parahaemolyticus biofilms formed on the glass surface, and could be directly observed using crystal violet staining. The reticular structure formed by the V. parahaemolyticus biofilm became denser with increasing cultivation time. The optimum parameters for V. parahaemolyticus biofilm formation with respect to biomass were determined using the plate reader. For a culture medium salinity of 3%, a rotation speed of 70 r/min, and an incubation time of 24 h, the number of bacteria in the mature V. parahaemolyticus biofilm was 2.56 × 107 CFU/cm2. Optimal biofilm detachment was obtained under pulsed ultrasound treatment(30-s sonication at intervals of 30 s) applying a frequency at 50 k Hz for four minutes, and the viability of bacteria was maintained under these conditions.
Crystal violet
Sonication
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The distribution of a carotene-containing yeast (CCY) in a biofilm formed by a small colony variant (SCV) of Pseudomonas aeruginosa PA01 was followed by confocal Raman microspectroscopy (CRM). SCV PA01 and CCY cells were distinguished by their spectral signatures, and the distribution of the overall biomass was monitored by the C–H bending or stretching signal. The distributions of total biomass, PA01, and CCY cells were compared at various times and positions within the biofilm. The distribution of the CCY was very heterogeneous. It was found in the water channels as well as in regions within biofilm colonies. Many of the yeast cells observed within the biofilm colonies under conditions of low or stopped flow were removed when medium was flowing, suggesting that the yeast was not held in the matrix as tightly as were the bacteria.
Raman microspectroscopy
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OBJECTIVE To investigate the effect of mesna on the formation of Pseudomonas aeruginosa biofilm,and study the effect of mesna on P.aeruginosa biofilm.METHODS The broth microdilution method was performed to determine the minimal inhibitory concentration of mesna to PAO1,then a biofilm model of Pseudomonas aeruginosa in vitro was established,the appearance of biofilm was detected by scanning electron microscope(SEM) to assess the effect of mesna on the formation of P.aeruginosa biofilm;the bacteria colony counts in biofilm was measured by agar plate after the biofilm was treated by mesna,biofilm structure was observed under confocal laser scanning microscope(CLSM),and the parameters of biofilm structure were analyzed through pictures from CLSM with image structure analyzer(ISA)software.RESULTS The MIC value against PAO1 was 10mg/mL for mesna.In the process of Pseudomonas aeruginosa biofilm formation,scanning electron microscope showed that the mucoid materials among bacteria was significantly reduced and the thickness of biofilm was decreased in mesna group.In comparison with normal saline group,viable counts in biofilms in the mesna treatment group were less than those in the saline group,and the high-dose group(4.06±0.12) had less positive effect than did the low-dose group(5.84±0.24)(P0.05).Confocal laser scanning microscope showed that the biofilm was thinner and more scattered than the saline control group.The results of ISA showed that with the treatment of mesna,biofilm was decreased in thickness,average diffusion distance(ADD) and textual entropy(TE) in comparison with the saline control group(P0.05),however areal porosity(AP) was increased(P0.05),and the high-doses group was more significant than the low-doses group(P0.05).CONCLUSION Mesna can inhibit the formation of P.aeruginosa biofilm and disrupt the structure of P.aeruginosa biofilm.
Mesna
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To establish the Escherichia coli biofilm model in vitro,the reference strain ATCC25922 was chosen for quantitative and qualitative analysis using modified crystal violet staining,rapid silver staining and scanning electron microscopy methods,and 249 clinical strains were tested for biofilm-forming ability analysis.Plate count method was used for growth curve mapping of Escherichia coli planktonic and biofilm bacteria.And tryptophan quantitative experiment was used to compare the impact of different culture conditions on extracellular matrix secretion of different biofilm-forming ability Escherichia coli.Rapid silver staining and scanning electron microscopy results showed that after biofilm formation,E.coli formed a special growth pattern which biofilm and planktonic bacteria co-located in the same living space.Different dilution of the bacterial inoculum on biofilm biomass change is not obvious(P0.05).The results showed that 249 E.coli were divided into four groups with strong,moderate,weak or absent biofilm-forming ability,accounting for 3.21%,18.88%,51.81% and 26.10%,respectively.
Crystal violet
Extracellular polymeric substance
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Objective To investigate the influence of different Mg2+ concentration on biofilm formation by mucoid Pseudomonas aeruginosa.Method Multifunction fluorometer was used to measure fluorescence of PA at the bottom of 96-well plate.The EPS of PA was stained with FITC-ConA and detected by fluorescence microscope.The biofilm was stained with SYTO9/PI,monitored by Confocal Laser Scanning Microscope(CLSM).Quantification of the effect was calculated by Image Structor Analyzer(ISA) sofeware.Result Two days group: after intervention with 1 mmol/L Mg2+,the fluorescence intensity was increased from 1845.67±45.39 to 2254.78±42.45,t=-9.96,P0.05;0.1 mmol/L Mg2+ could also increase the fluorescence intensity;Other time groups showed approximate tendency as two days group;the EPS in biofilm was significantly increased after intervention with Mg2+,which was visualized by fluorescence microscope,based on the FITC-ConA stain.The live bacteria in the biofilm increase and colony close-up after administration of Mg2+,when observed by CLSM,based on the SYTO9/PI stain.The quantitative data from ISA software showed that after administration of 1 mmol/L Mg2+,the depth of 6 day biofilm increase from(25.80±1.16)μm to(34.87±1.59)μm(t=-13.85,P0.05).Areal porosity decrease from 0.96±0.05 to 0.90±0.04(t= 2.48,P0.05),average diffusion distance increase from 1.54±0.15 to 1.92±0.16(t=5.23,P0.05),and textual entropy increase from 3.64±0.57 to 4.70±1.09(t=-2.6,P0.05).The 3 day biofilm showed the same tendency.Conclusion Magnesium can increase the initial attachment of PA and alter the subsequent biofilm formation and structure.
Fluorometer
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Objective:Isolate and screen a strain that has the ability to form biofilms from the samples of industrial spoilage with isothiazolinone,meanwhile research its preliminary characterization of biofilm formation.Methods:The streak plate and crystal violet staining methods were used to isolate and screen the biofilm-forming strain respectively.Morphological,physiological characteristics and 16S rDNA sequence analysis were adopted to determine the phylogenetic position of the isolated strain.The biofilms formed on the glass surface were observed under confocal scanning laser microscopy,including exopolysaccharides and live or dead cells.The influence of culture time,pH and different carbon sources on biofilm formation was also researched by crystal violet staining method in the microtiter plates.Results:A biofilm-forming strain was successfully isolated and named as BF-17,which was identified and clarified into Enterobacter cloacae.The highest biofilm biomass was found at the time of96 h or pH 5.0,respectively.After static cultured for 4 days,biofilms of BF-17 formed on the glass surface exhibited a representative morphological characteristics and structures of the typical biofilms.Meanwhile,the highest biofilm formation was formed in the M9 medium with the supplementation of α-lactose or D-fructose respectively.Whereas,addition of citric acid in M9 medium made the lowest biofilm formation.Conclusion:E.cloacae BF-17 shows a stable capacity of biofilm formation and can be used as a sample for further biofilm study.Moreover,the biofilm formation of BF-17 can be influenced by various external environment factors.
Crystal violet
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Microtiter plate
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Abstract The aim of this study was to evaluate the bioactivity and architecture of Candida albicans biofilms developed on the surface of poly(methyl methacrylate) (PMMA) resin. To do this, surface roughness (SR) and surface free energy of PMMA specimens were measured. Next, the biofilms of two different C. albicans strains (ATCC 90028 and SC5314) were allowed to develop on the PMMA surface and evaluated at 24, 48, and 72 h after adhesion. The bioactivity of the biofilms was measured by the XTT reduction assay. Biofilm topography was evaluated by scanning electron microscopy. Confocal microscopy was used to evaluate the architectural properties of bio‐volume, average thickness, biofilm roughness, surface area/volume ratio and the proportion of live/dead cells in the different biofilm development stages. SR and SFE had no influence on biofilm development. Each strain exhibited a different biofilm activity ( P < 0.001). Confocal images showed different architectures for the different biofilm development stages. We conclude that the main differences detected in biofilm bioactivity and architecture were related to the characteristics of each C . albicans strain and to biofilm development time. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010
Poly(N-isopropylacrylamide)
Strain (injury)
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