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 border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <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, &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&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/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/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.

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

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Immunology and Infection

8.2K Views.  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.

Video: Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...