The authors thank William Nance (University of Michigan) for help in formulating the biofilm growth protocols and John Battista (Fluxion, San Francisco, CA) for advice concerning technological issues relating to the Bioflux system. This work was supported by the National Institutes of Health (NIH: R21DE018820 to A. H. R.) and University of Michigan start-up funds to A. H. R.

The saliva collection protocol described herein was reviewed by the University of Michigan Institutional Review Board for Human Subject Research. 
NOTE: With regard to institutional reviews for human subject work of this type, prior arrangements and permissions should be garnered from the host institution. In particular, depending on the institution, IRB or ethics approval might need to be sought and approved before saliva collection from human volunteers can proceed. As an aid to preparing an application, a useful NIH algorithm/chart can be found here: <a href="http://grants1.nih.gov/grants/policy/hs/PrivateInfoOrBioSpecimensDecisionChart.pdf">http://grants1.nih.gov/grants/policy/hs/PrivateInfoOrBioSpecimensDecisionChart.pdf</a>
1. Preparation of Pooled Saliva for Use as an Environmentally Germane Growth Medium
<ol>
	<li>Recruit &#62;5 individuals for saliva donation. Do not take any identifying information, ensure no individuals will donate saliva if they are ill, or have taken oral antibiotics in the past 3 months, or have consumed food or liquid, with the exception of water, in the previous 2 hr before donation.</li>
	<li>Collect saliva in 50 ml plastic tubes. Pool the collected saliva in a plastic beaker, keeping it on ice. Do not use glass as polymers in the saliva will adhere to the internal glass surfaces.</li>
	<li>Add Dithiothreitol (DTT) to a final concentration of 2.5 mM from a 100x stock. (Stock is frozen in single-use aliquots at &#8211;20 &#176;C). Stir for 10 min in a plastic beaker on ice.</li>
	<li>In order to remove particulate matter, centrifuge the pooled saliva for 30 min at 17,500 x g.</li>
	<li>Dilute the saliva with 3 volumes of dH2O to give one-fourth concentrated saliva.</li>
	<li>Filter-sterilize saliva using 0.22 &#181;m polyethersulfone (PES), low protein binding filter. Use filter with large surface area, or use several small area filters. Keep saliva in a plastic container on ice while filtering.</li>
	<li>Freeze the pooled saliva at &#8211;80&#160;&#176;C in 50 ml plastic tubes until needed to grow bacteria. Each plastic tube is for one use only and should contain no more than 35 ml as each microfluidic well holds a maximum of 1.2 ml (1.2&#160;ml x 24 wells = 28.8 ml) and space is needed in each tube as saliva expands during freezing.</li>
	<li>For use, thaw the pooled saliva at room temperature. Once thawed, filter-sterilize once more (0.22 &#181;m polyethersulfone, low protein binding filter to remove any precipitates).</li>
</ol>
2. Preparation of Pooled Saliva for Use as an Inoculum
<ol>
	<li>Recruit &#62;5 individuals for saliva donation. Do not take any identifying information, ensure no individuals will donate saliva if they are ill, have taken oral antibiotics in the past 3 months, or have consumed food or liquid, with the exception of water, in the previous 2 hr before donation. Collect samples over 30 min.</li>
	<li>Collect saliva in 50 ml plastic tubes at room temperature and pool the collected saliva in a plastic beaker at room temperature.</li>
	<li>Dilute pooled saliva with autoclaved general reagent grade glycerol to yield a stock with a final ratio of 25% glycerol, 75% cell-containing saliva [CCS].</li>
	<li>Freeze saliva at &#8211;80&#160;&#176;C in 3 ml single-use aliquots until needed.</li>
	<li>When required to inoculate the microfluidic system, aliquots are thawed at room temperature and agitated gently on a vortexer for 5 sec before being pipetted into the microfluidic system as described in step 3 of the protocol, below.</li>
</ol>
3. Growth of Oral Multi-species Biofilms
<ol>
	<li>CFS pre-treatment
	<ol>
		<li>First coat the Bioflux microfluidic channels with CFS. Add 100 &#181;l of CFS to each outlet well. Using the Bioflux control software, select &#8220;manual&#8221; and set flow from channels &#8220;B1-B24&#8221; that are to be used.</li>
		<li>Next, set shear to 1.0 dyne/cm&#178; and start flow for 2 min at room temperature to ensure homogenous distribution of the CFS throughout the channel. Ensure there is fluid in each inlet channel to verify that CFS flowed through all channels evenly.</li>
		<li>Incubate plate at room temperature for 20 min.</li>
		<li>Remove the CFS/pretreat solution that remains in the outlet wells and transfer to the inlet wells. This total volume of 100 &#181;l will serve to balance against the pressure being applied to the inoculum from the outlet well.</li>
	</ol>
	</li>
	<li>Inoculation
	<ol>
		<li>To each outlet well, add 100 &#181;l of CCS inoculum. Place the microfluidic plate on the heat plate (temperature set at 37 &#176;C), select manual within the control software, and set flow from outlet wells to the inlet wells (i.e., reverse) at 1.0 dyne/cm&#178; for exactly 6 sec.</li>
		<li>Incubate the microfluidic plate at 37 &#176;C for 40 min to allow for initial adherence and growth of the bacteria in the inoculum.</li>
	</ol>
	</li>
	<li>Overnight Growth
	<ol>
		<li>For the inoculated channels being used, aspirate the waste inoculum from each of the outlet wells. Add up to 1 ml total volume of CFS into each of the inlet wells (can be done on top of existing CFS).</li>
		<li>Incubate the microfluidic plate at 37 &#176;C, select manual and set the program to run at 0.2 dyne/cm&#178; for 20 hr.</li>
	</ol>
	</li>
	<li>Stain prewash
	<ol>
		<li>Aspirate all fluid from the inlet and outlet wells and add 100 &#181;l of PBS (pH 7.4) to each of the inlet wells. Flow for 20 min at 0.2 dyne/cm&#178;.</li>
	</ol>
	</li>
	<li>Stain mixture addition
	<ol>
		<li>For cell viability staining make 100 &#181;l of stain mixture for each channel to be stained. Specifically, add 3 &#181;l of SYTO 9 and 3 &#181;l of propidium iodide per ml of PBS using commercial cell viability staining kit such as LIVE/DEAD. This generates a staining mixture containing 10 &#181;M SYTO 9 and 60 &#181;M propidium iodide.</li>
		<li>Aspirate the remaining PBS from the inlet wells and then add 100 &#181;l of the cell viability stain mixture to each inlet well. Set to flow at 0.2 dyne/cm2 and run the solution from inlet to outlet for 45 min at room temperature.</li>
	</ol>
	</li>
	<li>Post-staining wash
	<ol>
		<li>Aspirate the remaining stain in each of the inlet wells and add 100 &#181;l of PBS to each inlet well. Set to flow at 0.2 dyne/cm&#178; and run the PBS solution from inlet to outlet for 20 min at room temperature to remove any excess stain.</li>
	</ol>
	</li>
</ol>
4. Image Collection, 3D Rendering, and Image Analysis
<ol>
	<li>Use an inverted confocal laser scanning microscope (CLSM) to collect biofilm image data from the microfluidic system. Make sure that the CLSM is able to withstand the weight of the Bioflux plate, which can reach 150 g. 
	NOTE: Given the dimensions of the microfluidic channels, the need for sensitivity to discern biofilm structure, and the requirements to detect fluorescence signal of varying intensity, fit the CLSM with a 40X 1.25 NA objective lens or one with similar optical quality, magnification, and numerical aperture.</li>
	<li>Standardize the emission capture gates with gain and offset measurements being kept constant. Ensure that the laser power does not exceed 25% as this can result in photo-bleaching.</li>
	<li>Convert CLSM files from the Leica Image File type (LIF) to OME (Open Microscopy Environment) file type. This allows for greater ease of access and compatibility between software programs. Use commercially available software such as, IMARIS to convert files to this type.</li>
</ol>
3D Rendering for Images/Figures
<ol start="4">
	<li>Once converted to OME format, use commercially available software such as IMARIS to render the images in 3D. Perform this using a combination of &#8220;Easy3D&#8221; and &#8220;Surpass&#8221; options. 
	NOTE: Attention to background signal and thresholding should be made. The histogram function in IMARIS can be used to obtain the collection range and this should be kept constant between images being analyzed.</li>
	<li>Save images using the &#8220;snapshot&#8221; function and make careful consideration to image resolution before saving file types.</li>
	<li>Assemble images into figures using image editing software such as CorelDraw or Adobe Illustrator.</li>
</ol>
3D Image Analysis for Graphs and Tables
<ol start="7">
	<li>Use freely available IMAGEJ 23 and COMSTAT/COMSTAT2 38 for image analysis. Download the packages from http://rsb.info.nih.gov/ij/ and http://comstat.dk/, respectively. Have the most recent Java update installed. 
	NOTE: The software platform JAVA will need to be installed prior to the use of the image analysis software.
	<ol>
		<li>Use the IMAGEJ software with COMSTAT2 plugin to import the OME files for each biofilm image in &#8220;hyperstack&#8221; mode and view with &#8220;split channels&#8221;.</li>
		<li>Individually analyze the two channels (red/propidium-iodide/dead being channel 0, green/SYTO-9/LIVE being channel 1) using the &#8220;histogram&#8221; function of IMAGEJ. This function lists the total number of pixels of 8-bit OME files (shown as &#8220;count&#8221;) at each color intensity from 0 to 255 (shown as &#8220;value&#8221;), with 0 being pixels with no signal (background) and 255 being pixels with complete signal saturation.</li>
		<li>Export the data into a spreadsheet program and standardize all signal values by weighting each pixel by the corresponding signal intensity. This is performed by multiplying the total count at a given signal intensity by the numerical 8-bit value (0-255) of that signal intensity.</li>
		<li>Sum all weighted values, 0-255, for both channels for each biofilm image captured. Take the sum of the weighted values for both channels to determine the percent total signal from either channel. Do this to determine the relative ratio or percent red and percent green signal (i.e. the percent &#8220;DEAD&#8221; and percent &#8220;LIVE&#8221; for each treatment).</li>
		<li>Perform statistical analyses, for example using two-tailed Student&#39;s T-test modified for unequal variance. Values of p &#60;0.05 are considered significant and those values of p &#60;0.01 are considered highly significant.</li>
	</ol>
	</li>
</ol>
5. Harvesting Biofilm Cells for Culture-independent Analysis
<ol start="1">
	<li>Remove all spent and unused saliva from inlet and outlet wells. Wash the wells with 1 ml sterile distilled water three times.</li>
	<li>Add 100 &#181;l of sterile distilled water to the inlet wells and, using the software control interface, pass the sterile distilled water forward through the channels at &#62;8.0 dyne/cm2 (flow rate &#62;745 &#181;l/hr, shear of 800 sec-1) for at least 5 min. Repeat in the reverse direction for at least 5 min and then repeat the forward and reverse washing step process.</li>
	<li>Check by light or by epifluorescence microscopy (if cells were stained/labeled) for thorough biofilm removal (Figure 2). Collect the 100 &#181;l cell-suspension and store at -80&#160;&#176;C for culture-independent analysis. A cost-effective analysis of community composition can be achieved through using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) using the approach described in Nance et al. 20</li>
</ol>

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<li>Heydorn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (10), 2395-2407 (2000).</li>
<li>Nobbs, A. H., Lamont, R. J., Jenkinson, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Streptococcus+adherence+and+colonization.+Microbiology+and+molecular+biology+reviews%29%5BTitle%5D)+AND+%22MMBR%22%5BJournal%5D)">Streptococcus adherence and colonization. Microbiology and molecular biology reviews).</a> MMBR. 73 (3), 407-450 (2009).</li>
<li>Hope, C. K., Clements, D., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+the+spatial+distribution+of+viable+and+nonviable+bacteria+in+hydrated+microcosm+dental+plaques+by+viability+profiling%5BTitle%5D)+AND+%22Journal+of+applied+microbiology%22%5BJournal%5D)">Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling.</a> Journal of applied microbiology. 93 (3), 448-455 (2002).</li>
<li>Adams, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+laboratory+model+to+assess+the+removal+of+biofilm+from+interproximal+spaces+by+powered+tooth+brushing%5BTitle%5D)+AND+%22American+journal+of+dentistry+Spec+No+12B-17B%22%5BJournal%5D)">Development of a laboratory model to assess the removal of biofilm from interproximal spaces by powered tooth brushing.</a> American journal of dentistry Spec No 12B-17B. 15, 12-17 (2002).</li>
<li>Ledder, R. G., McBain, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+comparison+of+dentifrice+formulations+in+three+distinct+oral+microbiotas%5BTitle%5D)+AND+%22Archives+of+oral+biology%22%5BJournal%5D)">An in vitro comparison of dentifrice formulations in three distinct oral microbiotas.</a> Archives of oral biology. 57 (2), 139-147 (2012).</li>
</ol>

A. H. R. and N.S.J. have received research awards from a variety of sources such as the National Institutes of Health (NIDCR), Colgate-Palmolive (Piscataway, NJ) and the Society for Applied Microbiology to fund research studies in their lab over the past five years. All other authors: none to declare.

This methods paper highlights the basic steps required to setup and run a microfluidic system in a manner to allow for the development of oral multi-species biofilms derived from pooled human saliva and grown in filter-sterilized 25% pooled human saliva. Approaches to characterize the biofilm are given but it should be remembered that these described approaches are modifiable and additional technologies such as, for example, stains or labels can be introduced. As a matter of example, one could conceivably use labeled ant...

Biofilms are architecturally complex communities of bacteria that are aggregated on surfaces 1. These communities typically contain numerous species that interact with one another within the biofilm 2. Oral biofilms, the most visually conspicuous being dental plaque, are a persistent problem in humans and their uncontrolled development results in the generation of taxonomically diverse multi-species communities 3. The component bacteria of these diverse communities can be up to 1,000 times more resistant to antimicrobials than their free-floating (planktonic) counterparts 4-6. Failure to treat these oral biofilm communities, which can cause dental caries and periodontal disease, has resulted in a significant public health burden: over 500 million visits to the dentist office per annum in the US, and an approximately $108 billion to treat or prevent periodontal disease and dental caries 7.
&#8220;While many microbiologists advocate studying microbial behavior under natural conditions, few of them do so. This is because their morale for overcoming the difficulties is constantly sapped by the attractive ease of working with laboratory cultures.&#8221; &#8212;Smith 8.
At present, oral biofilm research is conducted using a variety of in vivo and in vitro approaches, each with their own advantages and disadvantages 9,10. In vitro approaches often use model biofilm systems that are relatively easy to set up but may lack clinical/real-world relevance 10,11. In vivo approaches typically rely upon animal model systems that may reproduce certain aspects of the human oral environment, but again suffer from limitations due to differences in anatomy, physiology, microbiology and immunology between animals and humans 12,13. It should be noted that oral biofilms can also be developed on enamel surfaces held in a stent within the mouths of human volunteers, but this approach is currently relatively costly and labor-intensive 14,15. Ultimately, novel agents or technologies to improve oral healthcare are tested in humans under controlled clinical trial conditions 11. At present, an often-used modus operandi for identifying and evaluating new oral healthcare agents is to first perform laboratory studies to discern potential efficacy, and then perform animal studies and &#8220;field trials&#8221; that employ clinicians to evaluate the success of the technology 9,16,17. Unfortunately, laboratory studies tend to rely on model systems that occupy a large footprint, are technologically challenging to use, and often contain simplified communities of one or at most a few species to derive potential real-world meaning 10,18. Given that dental plaque biofilms contain multiple species and form in a complex flowing salivary milieu, developing biofilms that contain one or a few species in artificial media is unlikely to generate communities that behave in a similar manner to those in a real-world scenario 10,19. To address the time, cost, training requirements, and the poor representative nature of laboratory model biofilm systems compared to the real-world environment, we recently developed a high throughput and environmentally germane biofilm system 20 (Figure 1). The system benefits from the use of cell-free pooled human saliva (CFS) as medium and untreated pooled human bacterial cell-containing saliva (CCS) as an inoculum. Uniquely, the system also combines microfluidic technology, a confocal laser scanning microscope, and culture-independent bacterial diversity analysis technology. Thus, the model system is environmentally germane (using saliva as an inoculum to grow multi-species biofilms at 37 &#176;C in filter-sterilized flowing saliva) and the oral biofilms contain species (including Streptococcus, Neisseria, Veillonella, and Porphyromonas species) in abundances representative of those found in early supragingival plaque 20.
When considering that this work describes the use of the newly developed model system, particular attention must be given to the amalgamation of a confocal laser scanning microscope (CLSM) microfluidics, and culture-independent diversity analysis technologies. The union of these technologies by our research group was intentional and not only adds a high-throughput capability to the newly developed model system, but also allows questions to be asked that could not be easily addressed before with other systems. Firstly, CLSM has distinct advantages over traditional microscopy as it allows for the three-dimensional analysis of biofilms. Often unappreciated, this is extremely important as biofilms are heterogeneous with respect to species composition and spatial position as well as the physiological conditions being imposed at different spatial locations within the biofilm 6,21. In concert with three-dimensional rendering software and image analysis software, the biofilm architecture, spatial relationships between component species, and extent of antimicrobial killing can be analyzed 22-24. Such abilities are not possible using standard transmitted light or epifluorescence microscopy. Next, microfluidics has garnered particular attention in the field of microbiology as it enables the study of biofilms under carefully controlled conditions (flow, temperature, pH, etc.) and only requires small volumes of liquid 25-27. As a point of comparison, growing an oral biofilm in human saliva within a flow cell model system (a system that is arguably considered the mainstay model for many oral biofilm studies) for 20 hr at a similar flow-rate and shear as that achieved in a microfluidic system requires at least 200 ml, as opposed to 800 &#181;l in the microfluidic device 28-31. Thus, a microfluidic model biofilm system enables the study of quantity-limited material under defined conditions. Finally, pyrosequencing technology has been optimized in the last decade to require only small amounts of material to perform a community analysis and is sufficiently versatile to control depth of sequencing to obtain the identity of even rare biofilm species. The use of this technology, such as bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), has allowed for pertinent questions concerning the ecology of biofilms to be addressed 32,33. Such questions imbued difficulties in the past when pyrosequencing was not available because of the time and costs required to create plasmid libraries and the complex technological and analytical steps required to derive data 33,34. Of course, a great advantage with culture-independent approaches, such as pyrosequencing, is that the bacterial species that cannot be grown in isolation within conventional laboratory media (i.e. viable but non-cultivable species) can be grown and identified within the model system and their relative abundance in the community quantified 35,36. To add perspective, as early as 1963, the late Sigmund Socransky estimated that approximately 50% of the bacteria in material isolated from the human oral gingival crevice could not be cultured using laboratory growth conditions 37.
The objective of this methods paper is to describe the approach to develop oral multi-species biofilms in a commercially available microfluidic (Bioflux) system under: (i) conditions representative of the human oral cavity and (ii) with a species composition and abundance that is comparable to supragingival plaque. Furthermore, using both freeware and commercial software, we highlight how basic biofilm architecture measures can be derived from CLSM data, with a focus on approaches to quantify biofilm biomass, roughness, and viability (based upon Live/Dead staining). Finally, the steps required to harvest biofilm material for diversity analysis by bTEFAP are described.

<table><tbody><tr><td>Falcon 50 ml Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Falcon 15 ml Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Dithiothreitol (White Crystals or Powder/Electrophoresis), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Sorval ultracentrifuge&amp;nbsp; (SS-34 compatible)</td><td>Thermoscientific</td><td>Unit-dependent</td><td></td></tr><tr><td>Thermo Scientific SS-34 Rotor&amp;nbsp;</td><td>Thermoscientific</td><td>28-020</td><td></td></tr><tr><td>Thermo Scientific Type 1 Reagent Grade Deionized Water</td><td>Thermo&amp;nbsp; Scientific Inc</td><td>23-290-065</td><td></td></tr><tr><td>Nalgene Rapid-Flow Filter Units and Bottle Top Filters, PES Membrane, Sterile.</td><td>VWR</td><td>73520-986&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific Inc</td><td>NC0542269</td><td></td></tr><tr><td>BioFlux microfluidic system</td><td>Fluxion</td><td>Bioflux 200 system</td><td></td></tr><tr><td>Bioflux 24-channel plate</td><td>Fluxion</td><td>910-0004</td><td></td></tr><tr><td>PBS (Gibco)</td><td>Thermo Fisher Scientific Inc</td><td>10010023</td><td></td></tr><tr><td>LIVE/DEAD stain (Invitrogen)&amp;nbsp;</td><td>Invitrogen</td><td>L7012</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Lecia</td><td>SPE or eqivalent system</td><td></td></tr><tr><td>Epifluorescence Microscope</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Pyrosequencing facilities</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Decon SaniHol 70 Ethanol Solution</td><td>Fisher Scientific</td><td>04-355-122</td><td></td></tr><tr><td>Ultra Low Temperature Freezer -80&amp;nbsp;&amp;deg;C</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Tips (20, 200, and 1,000&amp;nbsp;&amp;#956;l)</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Single Channel Variable Volume Pipettors (20, 200, 1,000 &amp;#956;l)</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt;</td></tr><tr><td>Bioflux dedicated software</td><td>Bioflux</td><td></td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Leica SPE</td><td>Leica</td><td></td><td></td></tr><tr><td>ImageJ</td><td>Freeware (http://imagej.nih.gov/ij/)</td><td></td><td></td></tr><tr><td>COMSTAT/COMSTAT 2</td><td>Freeware (http://www.comstat.dk/)</td><td></td><td></td></tr></tbody></table>

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.

3D Rendering of Biofilms
Representative results are shown in Figure 3. A useful tool in IMARIS software is the option to examine each slice of the collected biofilm stack and to combine them to create three-dimensional reconstructions. In addition, artificial shadowing effects can be added to help visually interpret three-dimensional structures. The rendered biofilms can be orientated in any direction with different magnifications to explore biofilm o...

Watch this Scientific Journal Video about Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms at JoVE.com

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.

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 ...

use-high-throughput-vitro-microfluidic-system-to-develop-oral-multi

Research

JoVE Journal

Bioengineering

13.5K Views.  The University of Michigan.  Het doel van deze methoden papier is het gebruik van een microfluïdische systeem te beschrijven voor de ontwikkeling van multi-species biofilms dat soort gewoonlijk geïdentificeerd in humaan supragingivale tandplaque bevatten. Methoden om biofilm architectuur, biofilm levensvatbaarheid, en een aanpak om te oogsten biofilm voor cultuur-afhankelijke of cultuur-onafhankelijke analyses zijn gemarkeerd beschrijven. 

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Immunology and Infection

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

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

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

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.

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

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

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.

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. ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Medicine

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

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

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