Funding for this study was provided by the National Institutes of Health/NIDCR grant 1R15DE019566-01A1. Dr. Jim Henthorn (OUHSC Flow and Image Cytometry Laboratory) is acknowledged for providing technical assistance with confocal laser scanning microscopy. Dr. Fernando Esteban Florez (Department of Dental Materials) is acknowledged for providing technical assistance during the filming of this video.

1. Preparation of Media and Reagents
<ol>
	<li>Make 300 ml overnight culture medium (THY; 3% Todd Hewitt Broth prepared according to manufacturer&#39;s instructions supplemented with 0.3% Yeast Extract in ultrapure water) and autoclave. Store at room temperature for up to 2 months.</li>
	<li>Make 1 L THY agar (3% Todd Hewitt Broth, 0.3% Yeast Extract, and 1.5% agar in ultrapure water), autoclave, and cool to 60 &#176;C before pouring into Petri dishes. Store at 4 &#176;C for up to 3 months.</li>
	<li>Make 150 ml biofilm growth medium (0.5X TY supplemented with 10 mM sucrose; 1.5% Tryptone, 0.5% Yeast Extract, and 10 mM sucrose in ultrapure water) and filter sterilize. Store at room temperature for up to 1 month.</li>
	<li>Make 1 L Phosphate Buffered Saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in ultrapure water) and autoclave. Store at room temperature for several months.</li>
	<li>Make 600 ml 10 mM Tris HCl buffer supplemented with 10 mM CaCl2 in ultrapure water (TC buffer; pH&#160;7.2) and autoclave. Store at room temperature for several months.</li>
	<li>Autoclave 1 L ultrapure water. Store at room temperature for several months.</li>
</ol>
2. Specimen Fabrication
<ol>
	<li>Fabricate disk-shaped specimens of Point 4 (PF) and TPH3 (TP) dental resin composites in a custom-made stainless steel mold in two increments. Each increment is light-cured for 40 sec using an LED light-curing unit. The two products have slightly different compositions and filler levels, and thereby produce different surface topographies upon which the biofilms will be grown.
	<ol>
		<li>Specimens of relevant substrates could be fabricated using established protocols from other disciplines in lieu of step 2.1.</li>
	</ol>
	</li>
	<li>Finish and polish specimens to an acceptable final surface finish, e.g.&#160;by using a semi-automated grinder-polisher. Rinse polished specimens with ultrapure water and dry using compressed air.</li>
	<li>Sterilize specimens using ethylene oxide gas or an alternate sterilization method.</li>
</ol>
3. Biofilm Growth
<ol>
	<li>Inoculate a single colony of S. mutans into 4 ml overnight culture medium (step 1.1). Incubate overnight at 37 &#176;C for 16-18 hr. The optical density (OD600) of the culture should be &#8805;0.9.
	<ol>
		<li>It is best to use a colony from plate cultures that have been passaged 2x from an original stock, as this results in more consistent biofilm growth.</li>
	</ol>
	</li>
	<li>Create a 1:100 dilution using the overnight culture (step 1.1) in 10 ml of biofilm growth medium (step 1.3).</li>
	<li>Transfer sterile resin composite specimens (e.g.&#160;PF, TP) to sterile 12-well plates.</li>
	<li>Add 2.5 ml of diluted culture medium (step 3.2) to wells for treatment (mouthwash) and control groups. Add 2.5 ml of sterile uninoculated biofilm growth medium to sterility control wells.</li>
	<li>Grow the biofilms under microaerophilic conditions. Place plates on a shaker at 100 rpm inside an incubator at 37 &#176;C for 24 hr.</li>
	<li>After 24 hr, aseptically aspirate medium from all wells and wash twice with sterile PBS (step 1.4; 2.5 ml/well), aspirating the PBS after each wash. Replenish 2.5 ml of fresh biofilm growth medium in each well and incubate for an additional 24 hr under identical conditions as the previous step, for total biofilm growth time of 48 hr.</li>
	<li>In lieu of the above steps, biofilms of other species could be grown on substrates using established protocols.</li>
</ol>
4. Antibacterial/Mouthwash Treatments
<ol>
	<li>Aspirate medium after biofilm growth is complete.</li>
	<li>Add 2.5 ml Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwash, or other treatment modality, in wells for the treatment groups, and add 2.5 ml sterile ultrapure water to the control group well. Immerse specimens for 60 sec, as per manufacturers&#39; instructions, while agitating plates on an orbital shaker at 150 rpm. Immediately aspirate both the mouthwash and ultrapure water from the wells.</li>
	<li>Wash the specimens 5x for 15 sec per wash with sterile ultrapure water on the orbital shaker at 150 rpm, aspirating water after each washing step.</li>
	<li>In lieu of the above steps, other antibacterial agents could be tested using established protocols.</li>
</ol>
5. Staining
<ol>
	<li>Prepare the stain dilutions for biofilm analysis.
	<ol>
		<li>Prepare a 5 mg/ml stock solution of Concanavalin A, Alexa Fluor 647 conjugate (AF) in 0.1 M sodium bicarbonate pH 8.3. Store at -20 &#176;C in single-use aliquots for several months because freezing and thawing is discouraged. Using this AF stock solution, prepare a 250 &#956;g/ml dilution in TC buffer.</li>
		<li>Prepare a 10 &#956;M dilution using the 5 mM Syto 9 stock solution (SY), as provided by the manufacturer, in TC buffer. Store the remaining SY stock solution at -20 &#176;C for several months.</li>
		<li>Prepare a 10x&#160;dilution using the 5,000x&#160;Sypro Red stock solution (SR), as provided by the manufacturer, in sterile ultrapure water. Store the remaining SR stock solution at -20 &#176;C for several months.</li>
	</ol>
	</li>
	<li>Wash all biofilms 2x with TC buffer (2.5 ml/well) using a hand-swirling motion, allowing the second wash to remain in the plate for 30 min at room temperature. Aspirate.</li>
	<li>Place a 50 &#956;l drop of AF stain on each biofilm specimen. Stain for 30 min at room temperature, protected from light. Wash 2x with TC buffer (2.5 ml/well), aspirating after each wash.</li>
	<li>Follow with a 50 &#956;l drop of SY stain on each biofilm specimen. Stain for 30 min at room temperature, protected from light. Wash 2x with sterile ultrapure water (2.5 ml/well), aspirating after each wash</li>
	<li>Finally, place a 50 &#956;l drop of SR stain on each biofilm specimen. Stain for 30 min at room temperature, protected from light. Wash 3x with sterile ultrapure water (2.5 ml/well), aspirating after each wash.</li>
	<li>Transfer the specimens to a 6-well plate, placing one specimen per well in 6 ml sterile ultrapure water to facilitate confocal microscopy.</li>
</ol>
6. Imaging Using Confocal Laser Scanning Microscopy
<ol>
	<li>Acquire images of each component (stain) within biofilms of the treatment and control groups using a confocal laser scanning microscope settings shown in Table 1. A 63X&#160;dipping lens with a 0.9 numerical aperture that has a working distance of 2.2 mm was used in this study. 
	<img alt="Table 1" fo:content-width="4.75in" src="/files/ftp_upload/50639/50639table1.jpg" /> 
	Table 1. Confocal Laser Scanning Microscope Settings.</li>
</ol>

<ol start="2">
	<li>Set the scan size to 250 &#956;m&#160;x&#160;250 &#956;m and a minimum pixel resolution of 512 x&#160;512 to acquire CLSM images at a resolution adequate for quantitative analysis.</li>
	<li>Use the SY stain to set the top and bottom points of the biofilm image stack, then set the z-step to be adequate for quantitative analysis (0.6 &#956;m for this study). Before collecting a scan, optimize the image and stain parameters (i.e.&#160;PMT voltage and offset) using the QLUT option. A color look-up table was used to assign pseudo-colors to each stain (green for SY, red for SR, and blue for AF) in order to make each biofilm component more distinguishable in the 3D reconstruction.</li>
	<li>Collect CLSM images at 400 Hz using sequential scanning in the &#34;between stacks&#34; mode to optimize capture of images of multiple stains within the same biofilm.</li>
</ol>
7. Biofilm Structural Analysis
<ol>
	<li>Analyze the CLSM images collected using image analysis software for biofilms. The following steps describe the use of ISA3D software12 to calculate the structural parameters of the biofilm images collected.
	<ol>
		<li>Copy CLSM image files for each biofilm to the Images folder, as specified in the software&#39;s manual. Ensure that the naming convention for CLSM files required by the software is followed. The software permits analysis of files within subfolders in batch mode, thus facilitating analysis of treatment and control group biofilms in the same run.</li>
		<li>Enter x-, y-, and z-axis dimensions of CLSM images in the dxy and dz fields of the main dialog box. Select threshold and distance mapping settings, as described in the software&#39;s manual (Otsu and Quasi-Euclidean were used for this study, respectively). Input a suitable name for the results file, and then run the program. A results file will be produced in the ISA folder.</li>
		<li>View the results file to obtain values for twenty 3D structural parameters12 such as biovolume and mean biofilm thickness (measured in this study) that quantify the 3D distribution of each component (stain) within the biofilm.</li>
	</ol>
	</li>
</ol>
8. Visualization of Biofilm Structure
<ol>
	<li>Create a reconstruction of overlaid components (stains) within each biofilm using image analysis software. The following steps describe the use of Volocity software to create 3D reconstructions.
	<ol>
		<li>Create a library for each biofilm by copying CLSM image files into the software, as described in the manual.</li>
		<li>Use the 3D Renderer menu option to produce a reconstructed 3D image from the CLSM images of each biofilm (HR Opacity format was used for this study).</li>
		<li>Rotate the 3D image to orient all of the biofilms in a similar manner with regard to the x-, y-, and z-coordinate axes. Capture a snapshot of the correctly oriented biofilm reconstruction, and then export the snapshot in TIFF, JPEG or other suitable format.</li>
	</ol>
	</li>
	<li>Perform visual analysis of the concurrent distribution of EPS, proteins and nucleic acid components within biofilms on the reconstructed images.</li>
	<li>Use Pearson&#39;s correlation coefficient and Manders&#39; overlap coefficient to perform a colocalization analysis of multiple biofilm components (colocalization was not measured in this study).</li>
</ol>
9. Statistical Analysis
<ol>
	<li>Use separate mixed models statistical analyses to compare mean values of structural parameters between mouthwash and control groups (&#945;=0.05). For this study, SAS software was used to perform the statistical analysis.</li>
</ol>

<ol>
	<li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: from the natural environment to infectious diseases.</a> Nat. Rev. Microbiol. 2, 95-108 (2004).</li><li>Yang, Y., Sreenivasan, P. K., Subramanyam, R., Cummins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+assessments+to+determine+the+effects+of+sugars+and+antimicrobials+on+a+polymicrobial+oral+biofilm.">Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm.</a> Appl. Environ. Microbiol. 72, 6734-6742 (2006).</li><li>Klein, M. I., Xiao, J., Heydorn, A., Koo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analytical+tool-box+for+comprehensive+biochemical,+structural+and+transcriptome+evaluation+of+oral+biofilms+mediated+by+mutans+streptococci.">An analytical tool-box for comprehensive biochemical, structural and transcriptome evaluation of oral biofilms mediated by mutans streptococci.</a> J. Vis. Exp. , (2011).</li><li>Conchello, J. A., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+microscopy.">Optical sectioning microscopy.</a> Nat. Methods. 2, 920-931 (2005).</li><li>Robinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+confocal+microscopy.">Principles of confocal microscopy.</a> Methods Cell Biol. 63, 89-106 (2001).</li><li>Neu, T. R., Kuhlicke, U., Lawrence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+fluorochromes+for+two-photon+laser+scanning+microscopy+of+biofilms.">Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms.</a> Appl. Environ. Microbiol. 68, 901-909 (2002).</li><li>Wood, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+intact+natural+human+plaque+biofilms+studied+by+confocal+laser+scanning+microscopy.">Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy.</a> J. Dental Res. 79, 21-27 (2000).</li><li>Chen, M. Y., Lee, D. J., Tay, J. H., Show, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+extracellular+polymeric+substances+and+cells+in+bioaggregates.">Staining of extracellular polymeric substances and cells in bioaggregates.</a> Appl. Microbiol. Biotechnol. 75, 467-474 (2007).</li><li>Zinchuk, V., Zinchuk, O., Okada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+multicolor+confocal+immunofluorescence+microscopy+images:+pushing+pixels+to+explore+biological+phenomena.">Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.</a> Acta Histochem. Cytochem. 40, 101-111 (2007).</li><li>Dunn, K. W., Kamocka, M. M., McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+evaluating+colocalization+in+biological+microscopy.">A practical guide to evaluating colocalization in biological microscopy.</a> Am. J. Physiol. Cell Physiol. 300, C723-C742 (2011).</li><li>Beyenal, H., Donovan, C., Lewandowski, Z., Harkin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+biofilm+structure+quantification.">Three-dimensional biofilm structure quantification.</a> J. Microbiol. Methods. 59, 395-413 (2004).</li></ol>

The authors declare that they have no competing financial interests. Production and Free Access to this article is sponsored by Leica Microsystems.

The acquisition of CLSM images has to be performed in the manner and format necessary for the quantification of biofilms and for colocalization analysis, such that the distribution of signal intensity in each image is a reliable representation of the distribution of each fluorochrome in the biofilm stack. The signal intensity should be distinguishable from noise and background10,11, unaffected by autofluorescence from the substrate, and having minimal bleed-through due to spectral interference between the fluorochromes used11. Noise is an inevitable limitation of fluorescence microscopy11, and image quality can sometimes be limited for technical or logistical reasons. Identification of fluorochromes with minimal spectral overlap is critical to the success of this method, but can be complicated. Furthermore, the quantification of multiple fluorochromes poses a multifactorial problem. Therefore, it is not surprising that very few investigations have successfully quantified the concurrent distribution of more than two components within the structures of biofilms.
The objective of this study was to report a methodology consisting of distinct but interconnected steps for the quantification and comparison of the concurrent 3-dimensional distributions of three cellular and extracellular components within biofilms. The biofilm growth assay described permits biofilm growth on relevant substrates and produces biofilm structures that are reproducible, as demonstrated by the results reported in Table 1. The combination of novel simultaneous staining of EPS, proteins and nucleic acid components with the measurement of 3D biofilm structural parameters results in quantifiable distributions of components within biofilms. Qualitative visual analysis of the concurrent distribution of EPS, proteins and nucleic acid within the biofilms is possible via the reconstruction of overlaid stains within each biofilm. The ISA3D software12 contains several unique structural parameters such as fractal dimension, homogeneity, and biofilm roughness coefficient in addition to traditionally-measured parameters such as porosity and biofilm thickness to enhance quantification of the 3D structures of biofilms. Statistical analysis of the biofilm structural parameters facilitates relevant comparisons of biofilms under specific experimental conditions such as antibacterial/antifouling efficacy (e.g.&#160;after treatment with mouthwashes) or over time (temporal effects).
This methodology efficiently and successfully quantified and compared the concurrent 3D distributions of three major components within S. mutans biofilms that were grown on relevant substrates, thus overcoming two challenges to the simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial treatments or antifouling measures against multiple biofilm components, as shown using mouthwashes in this study. Furthermore, the majority of the protocols described above can be replaced with protocols for the fabrication of relevant substrates and biofilm growth assays of relevant microorganisms. Therefore, this method has broad application because it facilitates comparison of 3D structures/architecture of in vitro and in vivo biofilms in a variety of disciplines including the medical, dental, geological and marine sciences.

Biofilms are structured microbial communities that are encapsulated in a self-produced extracellular matrix, and are attached to biological or inert surfaces1. Biofilms represent a common lifestyle for many bacteria, and form by transitioning in stages from free-floating (planktonic) cells to complex multispecies communities. The inherent resistance of biofilms to antimicrobial agents is at the root of many persistent and chronic bacterial infections1,2, as demonstrated by oral biofilms (dental plaque). Cariogenic microorganisms such as mutans streptococci process sucrose and other carbohydrates to produce an extracellular matrix and generate acids that can demineralize tooth structure and cause dental caries. Most biofilm matrices are biopolymers consisting of cellular and extracellular components such as exopolysaccharides (EPS), proteins, and nucleic acids3,4.
Confocal laser scanning microscopy (CLSM), the most widely used technique for fluorescence imaging, has radically transformed optical imaging in biology because it has the ability to collect 3D images of hydrated biological structures without fixation5,6,7. This nondestructive technique involves collecting images of thin sections within a region of interest on the specimen in such a manner that the contribution of out-of-focus light is removed. The quality and resolution of images captured by CLSM is beyond what is achievable using widefield fluorescence microscopy. One major drawback of CLSM is that scanning of images occurs at a slower rate than with widefield microscopy techniques, in which entire images are collected simultaneously5. However, with a widening selection of fluorochromes, lasers, and filters, CLSM has become one of the prevailing techniques for multispectral imaging5,7.
Previous studies have shown CLSM to be a useful tool for examining the structure or architecture of biofilms by using one or two fluorescent tags or stains to provide a better understanding of the distribution of EPS and cells within biofilms, and especially within the extracellular matrix7,8. In theory, fluorescent staining/labeling of multiple components is desirable for exploring the detailed structure and colocalization of cellular and extracellular components within biofilms. However, concurrent analysis of various components within biofilms can be challenging because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Colocalization using multiple fluorochromes requires the use of highly specific stains with minimal spectral interference to avoid any bleed-through effects, which occurs when two fluorochromes have significant overlap in their spectral peak, causing one to be more strongly excited than the other9. Ideally, fluorochromes having excitation spectra that do not overlap would provide the best results, however it is very difficult to find stains that meet this criterion. Instead, the selection of stains is optimized by choosing fluorochromes whose emission spectra have minimal overlap, allowing the stains to be viewed one by one within a limited observation wavelength band9.
Superimposition of fluorescence images is probably the most widely used method for evaluating concurrent distribution of fluorochromes. Colocalization of the various components appears as an overlap of different colors through multiple channels created by the fluorochromes being examined10. The tools for displaying multiple-channel fluorescence images as merged color images are available in most CLSM software and biological image analysis software. Although superimposition of images is useful for spatial evaluation of colocalization, the images can only be examined qualitatively by visual analysis. This provides a limited amount of information, as these representations are generally not helpful for quantifying colocalization under different experimental conditions nor do they determine whether the colocalization exceeds random coincidence11. Very few investigations so far have used quantitative methods to analyze the 3-dimensional structure of biofilms and biofilm components, and even fewer have quantified the effect of antibacterial treatments or antifouling measures on biofilm components.
The objective of this study was to report a methodology for quantification and comparison of the concurrent 3-dimensional distributions of three cellular and extracellular components of biofilms. The method consists of distinct but interconnected steps involving biofilm growth, staining, CLSM imaging of biofilms, biofilm structural analysis and visualization, and statistical analysis of structural parameters. The biofilm growth assay permits biofilm growth on relevant substrates, and produces biofilm structures that are reproducible. The combination of novel simultaneous staining of EPS, proteins and nucleic acid components with the measurement of 3D biofilm structural parameters results in quantifiable distributions of components within biofilms. Statistical analysis of the biofilm structural parameters facilitates evaluation of biofilms under specific experimental conditions (e.g.&#160;after treatment with mouthwashes), as will be described in the next section.

<table><tbody><tr><td>Bacto Agar</td><td>Becton, Dickinson and Company</td><td>214010</td><td></td></tr><tr><td>Bacto Todd Hewitt Broth</td><td>Becton, Dickinson and Company</td><td>249240</td><td></td></tr><tr><td>Yeast Extract, Granulated</td><td>EMD Millipore</td><td>1.03753.0500</td><td></td></tr><tr><td>Bacto Tryptone</td><td>Becton, Dickinson and Company</td><td>211705</td><td></td></tr><tr><td>OmniPur Sucrose</td><td>EMD Millipore</td><td>8510</td><td></td></tr><tr><td>Potassium Chloride, ACS reagent, 99.0-100.5%</td><td>Sigma-Aldrich</td><td>P3911-500G</td><td></td></tr><tr><td>Potassium Phosphate, monobasic, &amp;ge;99.0%, ACS reagent</td><td>Sigma-Aldrich</td><td>P0662-500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S9888-500G</td><td></td></tr><tr><td>Sodium Phosphate, Monobasic, Monohydrate</td><td>EMD Millipore</td><td>SX0710-1</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane, 99.8+%, ACS reagent</td><td>Sigma-Aldrich</td><td>252859-500G</td><td></td></tr><tr><td>Concanavalin A, Alexa Fluor 647 Conjugate</td><td>Invitrogen</td><td>C21421</td><td></td></tr><tr><td>Syto 9</td><td>Invitrogen</td><td>S34854</td><td></td></tr><tr><td>Sypro Red</td><td>Invitrogen</td><td>S12012 or S6653</td><td></td></tr><tr><td>Biot&amp;egrave;ne PBF Oral Rinse</td><td>GlaxoSmithKline</td><td>N/A</td><td></td></tr><tr><td>Listerine Total Care</td><td>McNeil-PPC, Inc.</td><td>N/A</td><td></td></tr></tbody></table>

concurrent quantification of cellular and extracellular components of biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

Representative results for the treatments (mouthwashes) and untreated control group are shown in Table 2, and Figures 1 and 2. Table 2&#160;displays the mean and standard deviation values of biofilm structural parameters biovolume (&#956;m3) and mean biofilm thickness (&#956;m) that were calculated using ISA3D software. Structural parameters of biofilms treated with mouthwash that differed significantly from those of biofilms in the control group (p&#60;0.05) have mean and standard deviation values highlighted in red. The results of the mixed models statistical analyses demonstrated that mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas mouthwash LTO did not produce significant differences (p&#62;0.05) on either resin composite. The results clearly show that the cellular and extracellular biofilm components remaining after the two mouthwash treatments differed. It should also be noted that the S. mutans biofilms grown on the two substrates (PF and TP) differed in 3D structure even though they were grown under similar conditions and both substrates were polished with similar abrasives.
The concurrent distribution of EPS, proteins and nucleic acid within biofilms can be visualized via the 3D reconstructions generated using Volocity software. Figures 1 and 2 demonstrate representative reconstructions of control group biofilms grown on PF and TP resin composites, respectively. The blue stain represents EPS within S. mutans biofilms, the green stain demonstrates nucleic acids, and the red stain shows proteins. The intervening space may be occupied by water or other nonfluorescently labeled biofilms components.
<img alt="Figure 1" fo:content-width="4.75in" fo:src="/files/ftp_upload/50639/50639fig1highres.jpg" src="/files/ftp_upload/50639/50639fig1.jpg" /> 
Figure 1. A representative 3D reconstruction of S. mutans biofilm grown on PF resin composite in the control group (not treated with mouthwash). Concurrent overlay of the three stains within a single biofilm permits the simultaneous visualization of EPS (blue stain), nucleic acid (green stain), and protein (red stain) components within S. mutans biofilms. (1 Unit = 24 &#956;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50639/50639fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="4.75in" fo:src="/files/ftp_upload/50639/50639fig2highres.jpg" src="/files/ftp_upload/50639/50639fig2.jpg" /> 
Figure 2. A representative 3D reconstruction of S. mutans biofilm grown on TP resin composite in the control group (not treated with mouthwash). Concurrent overlay of the three stains within a single biofilm permits the simultaneous visualization of EPS (blue stain), nucleic acid (green stain), and protein (red stain) components within S. mutans biofilms. (1 Unit = 24 &#956;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50639/50639fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1">
	<tbody>
		<tr>
			<td>Resin composite</td>
			<td></td>
			<td colspan="2">Point 4</td>
			<td colspan="2">TPH3</td>
		</tr>
		<tr>
			<td>Mouthwash</td>
			<td>Component</td>
			<td>BV (&#956;m3)</td>
			<td>MT (&#956;m)</td>
			<td>BV (&#956;m3)</td>
			<td>MT (&#956;m)</td>
		</tr>
		<tr>
			<td>Biot&#232;ne PBF</td>
			<td>Nucleic Acids</td>
			<td>279,517&#177;53,291</td>
			<td>9.32&#177;2.80</td>
			<td>195,033&#177;42,014</td>
			<td>7.45&#177;3.70</td>
		</tr>
		<tr>
			<td></td>
			<td>EPS</td>
			<td>344,902&#177;56,386</td>
			<td>35.22&#177;17.19</td>
			<td>197,840&#177;62,351</td>
			<td>9.83&#177;7.26</td>
		</tr>
		<tr>
			<td></td>
			<td>Proteins</td>
			<td>298,796&#177;62,868</td>
			<td>54.21&#177;21.65</td>
			<td>216,033&#177;66,654</td>
			<td>24.33&#177;39.64</td>
		</tr>
		<tr>
			<td>Listerine Total Care</td>
			<td>Nucleic Acids</td>
			<td>355,707&#177;110,444</td>
			<td>26.45&#177;14.21</td>
			<td>273,296&#177;47,323</td>
			<td>13.43&#177;2.89</td>
		</tr>
		<tr>
			<td></td>
			<td>EPS</td>
			<td>494,099&#177;180,592</td>
			<td>64.90&#177; 26.68</td>
			<td>329,150&#177;47,145</td>
			<td>34.35&#177;30.32</td>
		</tr>
		<tr>
			<td></td>
			<td>Proteins</td>
			<td>348,416&#177;161,316</td>
			<td>58.68&#177;47.28</td>
			<td>303,150&#177;54,705</td>
			<td>34.18&#177;41.46</td>
		</tr>
		<tr>
			<td>Control (untreated)</td>
			<td>Nucleic Acids</td>
			<td>388,375&#177;42,152</td>
			<td>51.15&#177;40.66</td>
			<td>327,809&#177;39,400</td>
			<td>17.08&#177;1.65</td>
		</tr>
		<tr>
			<td></td>
			<td>EPS</td>
			<td>660,448&#177;173,197</td>
			<td>91.37&#177;74.84</td>
			<td>363,850&#177;67,612</td>
			<td>28.33&#177; 15.07</td>
		</tr>
		<tr>
			<td></td>
			<td>Proteins</td>
			<td>517,274&#177;119,475</td>
			<td>127.96&#177;73.84</td>
			<td>353,161&#177;56,518</td>
			<td>21.17&#177;4.41</td>
		</tr>
	</tbody>
</table>
Table 2. Mean and standard deviation values of Biovolume (BV) and Mean biofilm thickness (MT) of biofilms treated with BIO or LTO, or left untreated (control group). Structural parameters of biofilms treated with mouthwashes that differed significantly from those of biofilms in the control group (p&#60;0.05) have mean and standard deviation values highlighted in red.

Watch this Scientific Journal Video about Concurrent Quantification of Cellular and Extracellular Components of Biofilms at JoVE.com

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

concurrent-quantification-cellular-extracellular-components

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.3K 次观看.  University of Oklahoma Health Sciences Center. A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

视频: Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

科研

免疫与感染

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

环境科学

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

生物工程

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under different growth conditions and after treatment with matrix modifying molecules. To better quantitate the material properties of 3D biofilms, an experimental and computational workflow was developed and applied to examine differences between Enterococcus faecalis, Salmonella enterica serotype Typhimurium and Escherichia coli biofilms as well as the role of the amyloid curli in confirming rigidity to Enterobacteriaceae biofilms. The spatio-temporal dynamics of 1 &#181;m carboxylate beads in biofilms were tracked in 20 &#181;m 3D biofilms over 20 minutes. The 4D image stacks were processed using the Mosaic plugin in ImageJ to produce 3D trajectory data of bead movement. This trajectory data was analyzed with a newly developed Bead Evaluator toolbox, where movement data, including trajectory lifespans, bead velocities, cell densities along trajectories, and bounding box information were computed and stored in csv-files. This paper presents the workflow from experimental setup and image recording to bead trajectory computation and analysis. The structure of curli-containing biofilms resulted in more stable bead interactions and less bead movement than in curli-mutant and Enterococcal biofilms. Bead movement did not appear strongly dependent on cell density when measuring the bead velocity and trajectory bounding box volume, supporting the hypothesis that other material properties of the biofilms control the bead dynamics. This technique is widely applicable to quantitating differences in biofilms of different matrix compositions as well as biofilms before and after matrix-modifying treatments.

We describe a method for analyzing and quantifying the movement pattern of 1 &#181;m carboxylate beads through heterogeneous bacterial biofilms. Comparison of the movement patterns can be used to quantitate differences in material properties of biofilms.

Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under ...

细胞和生物膜细胞外成分的定量并发

摘要

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此视频中的章节

铜绿假单胞菌和酿酒酵母流细胞生物膜

在体外生物膜的形成以及8商会幻灯片

流式细胞仪对水生生物膜的表征

使用高通量的

方法表征的共同发展生物膜和生境异质性

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

用两相循环流系统实时成像和定量真菌生物膜的研究

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

细胞和生物膜细胞外成分的定量并发

摘要

探索更多视频

此视频中的章节

铜绿假单胞菌和酿酒酵母流细胞生物膜

在体外生物膜的形成以及8商会幻灯片

流式细胞仪对水生生物膜的表征

使用高通量的

方法表征的共同发展生物膜和生境异质性

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

用两相循环流系统实时成像和定量真菌生物膜的研究

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响