This work was supported by NHI R01 grant GM59690 awarded to KLV.

1. Initial Characterization and Considerations
<ol>
 <li>Biofilm formation is generally influenced by cell density and growth rate. Therefore, it is necessary to determine the growth rate (increase in optical density (OD) over time) and yield (final cell number) of the strain(s) of interest by performing simple growth curve and cell plating assays. Defects in growth, or a lack of correlation between OD and cell number, must be taken into consideration when interpreting results from spotting experiments.</li>
 <li>Include the appropriate positive and negative controls on the same plate when assessing wrinkled colony formation, as minor plate-to-plate variation may impact biofilm development.</li>
 <li>Identify the best conditions for spotting, i.e., those that reveal the most distinct differences between the control and mutant(s) of interest. Grow strains in liquid culture under various conditions, such as different media or temperatures, and to different stages of growth (exponential or stationary phase) prior to spotting. Spot 10 &mu;l of culture at various cell densities onto the appropriate media, and incubate at the desired temperature until colony morphologies become apparent.</li>
 <li>The end-point assay involves assessment of wrinkled colony formation at a predetermined time point after spotting (i.e., 48 hours). This assessment is useful for strains that exhibit (or are proposed to exhibit) severe defects in biofilm formation.</li>
 <li>The time course assay assesses wrinkled colony formation over a period of time (i.e., every hour) post-spotting. This assay permits a semi-quantitative assessment of biofilm formation by allowing the determination of the start of wrinkled colony formation and pattern development over a period of time. The duration of the experiment and number of collection points for a given time course should be determined in preliminary experiments.</li>
 <li>To easily keep track of the time post-spotting, set a timer to count up.</li>
</ol>
2. Microscopic Assessment of Wrinkled Colony Morphology in V. fischeri
<ol>
 <li>Inoculate V. fischeri cells into 5 ml of LB-Salt (LBS) medium 13 (1% [w/v] tryptone, 0.5% [w/v] yeast extract, 2% [w/v] sodium chloride, 50 mM Tris-HCl [pH 7.5]) containing any necessary antibiotics and incubate, with shaking, overnight at 28 &deg;C. In the morning, subculture the cells with a 1:100 dilution into 5 ml of fresh LBS medium and incubate under the same conditions until the cells have reached the desired OD600 (e.g., OD600=0.2 or 0.5).</li>
 <li>Pipette 1 ml of culture into a microfuge tube and centrifuge in a microfuge set at maximum speed for one minute. Remove the supernatant by aspiration. Wash the cells to remove residual media and extracellular components by resuspending the pellet in 1 ml of sterile 70% artificial seawater (ASW) (35 mM MgSO4-7H2O, 7 mM CaCl2-2H2O, 210 mM NaCl, 7 mM KCl) and repeating the centrifugation. Resuspend the washed pellet in 1 ml of 70% ASW.</li>
 <li>Make sure that each sample contains the same number of cells, as estimated by the OD at 600 nm. Make any necessary adjustments by diluting more concentrated samples with additional 70% ASW. In preliminary experiments, spot cultures at various initial OD values to determine an optimal starting OD for a given set of strains or conditions. The best results are obtained for V. fischeri when spots are generated from cultures with an OD of approximately 0.2.</li>
 <li>Spot 10 &mu;l of the washed cells onto LBS plates containing any necessary antibiotics. When spotting a culture onto a plate, make sure to steady the pipette (with your finger) just above the agar surface. Spot vertically, not at an angle, and eject the liquid slowly for uniform distribution of the spot. Typically each strain is spotted once per plate (up to 6 spots per plate), with multiple plates (2-3) per experiment.</li>
 <li>To ensure that the spotted culture remains evenly distributed, allow the spot to dry before moving the plate to the incubator. Invert the plates and incubate them at 28 &deg;C.</li>
 <li>Monitor the morphology of the growing spot hourly beginning at 12-15 hours following inoculation. We use a dissecting scope (Zeiss stemi 2000-C) with a camera attachment (ProgRES C10PLUS) and the ProgRes CapturePro and ImageJ software programs to observe and document colony morphology and to assess the start and progression of wrinkled colony formation.</li>
 <li>For the specific setup listed in step 2.5, the spots are illuminated from underneath through a transparent glass stage with a CL 1500 EC cold light source, while the images are captured from above. This setup is optimal for imaging wrinkled colony development because V. fischeri colonies (spots) are translucent.</li>
 <li>In the absence of the specific equipment listed in step 2.5, monitor wrinkled colony morphology using any dissecting microscope that allows for full colony viewing and has an adjustable light source and, ideally, an attached camera. If necessary, a digital camera can also be utilized in the absence of an attached camera, but this is not optimal.</li>
 <li>To best visualize wrinkled colony development, it is necessary to adjust both the lighting intensity and angle of reflection underneath the bacterial colonies such that the three-dimensional morphology of the developing biofilms can be discerned. Determine the optimal lighting conditions that provide the strongest contrast between the spotted colony and the surrounding agar background, such that the architecture (wrinkles) of the colony is clearly distinguishable.</li>
 <li>In some cases, the color of the spotted colony must also be taken into consideration, as alterations in colony color can occur during biofilm formation. Adjust the intensity and angle of the lighting source to reveal these subtle changes in coloration. Once the appropriate settings are achieved, maintain them for the duration of the experiment.</li>
 <li>Note how much time elapses from the time of inoculation to the time at which wrinkled colony formation initiates for each strain or condition. Define the start of wrinkled colony formation as the time point at which the formation of patterning and 3D structures (i.e., striations forming at the outer edge or 'ripples' occurring in the center) is first apparent. Mutant cells may exhibit decreased or increased time to the start of wrinkled colony formation (e.g., Fig. 2).</li>
 <li>Document the start and development of wrinkled colony formation by capturing appropriate digital images. It is important to use the same magnification when collecting images throughout the experiment. Switch the view from the eyepiece of the microscope to the computer screen using the lever located on the back of the camera. When switching between eyepiece and computer screen viewing, adjust the view and focus accordingly.</li>
 <li>Before imaging spotted cultures, the lid of the Petri plate is typically removed to provide the clearest image. However, this is an optional step, as spotted cultures can be imaged through the lid using the setup outlined in steps 2.5 and 2.6.</li>
 <li>At each time point following the start of wrinkled colony formation, note the pattern of wrinkled colony development. The architecture may develop from the inside out, or the outside in. This assessment provides a mechanism to distinguish the biofilms formed by different strains or under different conditions.</li>
 <li>Measure the diameter of the developing colony at each time point. This can be done manually, or digitally using an associated software program. If done digitally, use the ProgRes CapturePro software program and first calibrate the scale bar to the specific magnification used in the experiment. Include the scale bar in each image captured.</li>
 <li>To calculate the colony diameter using the ImageJ software program, open each image file. Standardize the scale bar as follows: Select the "Straight line option" from the toolbar. Overlay the embedded scale bar with a straight line of the same length and width. Select "Set Scale" from the tab labeled "Analyze." In the "known distance" box, insert the corresponding length (as determined from the original scale bar; i.e., 2 mm) and select "OK".</li>
 <li>Use the "Straight line" option to insert a horizontal line across the spot. Under the Analyze tab, select "Measure". In the new Results window, the calculated length will be provided. Obtain a second measurement by inserting a perpendicular line across the spot. Re-calculate the diameter. Record the average of the two measurements. Graph the average diameter for each spot at each time point using a software program such as Excel.</li>
 <li>The end of the experiment occurs either at a specified time point or when there is no further development of the biofilm. Once the end is reached, consolidate the images into a figure(s) to visualize the pattern development over time using a software program such as Powerpoint.</li>
</ol>
3. Representative Results
In these experiments we used V. fischeri as a model organism to study biofilm formation by evaluating the development of wrinkled colonies on a solid agar surface. Biofilm-producing strains of V. fischeri form colonies with extensive 3D architecture within 40 h (Fig. 1) 8,9,14. When examined over a time course, it becomes apparent that wrinkled colony formation by the control strain initiates as early as about 12 h post-inoculation (depending on the specific conditions) (Fig. 2) 9. In contrast, biofilm formation by a representative mutant is delayed by approximately 4 h, not initiating until approximately 16 h post-inoculation 9. Repeats of these experiments suggested that the timing was relatively consistent, making this assessment semi-quantitative 9. A second semi-quantitative measure of biofilm formation makes use of the change in diameter of the developing colony over time. As shown in Fig. 3A, a representative biofilm-competent strain forms colonies that increase in complexity and diameter relative to a representative strain that does not form biofilms. Careful measurements over time of the diameters of the colonies formed by these two strains revealed that the size of the biofilm-competent colonies increased at a greater rate than the biofilm-negative colonies, and at the end time point the two differed by almost 2-fold (Fig 3B). Thus, although images of a representative late time or "end-point" colony morphology frequently are shown in the literature, additional, semi-quantitative data can be collected that will permit a better understanding of the biofilm defect.
<img alt="Figure 1" src="/files/ftp_upload/4035/4035fig1.jpg" /> 
Figure 1. End-point assay. This figure is an example of an end-point assay using representative non-biofilm-forming (left) and biofilm-forming (right) strains of V. fischeri. These images were collected at 40 h post-spotting. These images were generated with wild-type V. fischeri containing a vector control (non-biofilm-forming) or an rscS overexpressing plasmid (biofilm-forming) and were part of the data set collected for Morris et al., 2011.
<img alt="Figure 2" src="/files/ftp_upload/4035/4035fig2.jpg" /> 
Figure 2. Time-course assay. The upper panel contains representative images of biofilm formation by a biofilm-competent control strain of V. fischeri over a selected time course. Initiation of biofilm formation is evident at 12 h. The lower panel contains representative images of a mutant strain of V. fischeri that exhibits a delay (4 h) in the start of wrinkled colony formation over time, with biofilm formation initiating at 16 h post-inoculation. Note that at 40 hours the strains look similar in the intensity and patterning of biofilm formation, while subtle differences between these strains are only observed at the earlier time points. These images were generated with rscS overexpressing wild-type and sypE mutant V. fischeri cells and were part of the data set collected for Morris et al., 2011.
<img alt="Figure 3" src="/files/ftp_upload/4035/4035fig3.jpg" /> 
Figure 3. Colony diameter as a semi-quantitative analysis of biofilm formation. (A) Time course of biofilm formation by representative biofilm-forming (upper panel) and non-biofilm-forming (lower panel) strains of V. fischeri. Note that the biofilm-forming strain exhibits a greater increase in diameter over time relative to the non-biofilm forming strain. These images were generated with wild-type V. fischeri containing an rscS overexpressing plasmid (biofilm-forming) or a vector control (non-biofilm-forming) and were part of the data set collected for Morris et al., 2011. (B) A graphical representation of the increase in colony diameter over time by the two strains in panel A. These data were generated using the ImageJ and Excel software as outlined in the protocol.

<ol>
	<li>Donlan, R. M., Costerton, 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=Biofilms:+survival+mechanisms+of+clinically+relevant+microorganisms.">Biofilms: survival mechanisms of clinically relevant microorganisms.</a> Clin. Microbiol. Rev. 15, 167-167 (2002).</li><li>Stoodley, P., Sauer, K., Davies, D. G., Costerton, 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=Biofilms+as+complex+differentiated+communities.">Biofilms as complex differentiated communities.</a> Annu. Rev. Microbiol. 56, 187 (2002).</li><li>Branda, S. S., Vik, S., Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+the+matrix+revisited.">Biofilms: the matrix revisited.</a> Trends Microbiol. 13, 20 (2005).</li><li>Branda, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruiting+body+formation+by+Bacillus+subtilis.">Fruiting body formation by Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11621 (2001).</li><li>Beyhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+rugosity+and+biofilm+formation+in+Vibrio+cholerae:+comparison+of+VpsT+and+VpsR+regulons+and+epistasis+analysis+of+vpsT,+vpsR,+and+hapR.">Regulation of rugosity and biofilm formation in Vibrio cholerae: comparison of VpsT and VpsR regulons and epistasis analysis of vpsT, vpsR, and hapR.</a> J. Bacteriol. 189, 388 (2007).</li><li>Enos-Berlage, J. L., Guvener, Z. T., Keenan, C. E., McCarter, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+determinants+of+biofilm+development+of+opaque+and+translucent+Vibrio+parahaemolyticus.">Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus.</a> Mol. Microbiol. 55, 1160 (2005).</li><li>Merritt, J. H., Brothers, K. M., Kuchma, S. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SadC+reciprocally+influences+biofilm+formation+and+swarming+motility+via+modulation+of+exopolysaccharide+production+and+flagellar+function.">SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function.</a> J. Bacteriol. 189, 8154-8154 (2007).</li><li>Yip, E. S., Geszvain, K., DeLoney-Marino, C. R., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+symbiosis+regulator+rscS+controls+the+syp+gene+locus,+biofilm+formation+and+symbiotic+aggregation+by+Vibrio+fischeri.">The symbiosis regulator rscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri.</a> Mol. Microbiol. 62, 1586-1586 (2006).</li><li>Morris, A. R., Darnell, C. L., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+a+novel+response+regulator+is+necessary+for+biofilm+formation+and+host+colonization+by+Vibrio+fischeri.">Inactivation of a novel response regulator is necessary for biofilm formation and host colonization by Vibrio fischeri.</a> Mol. Microbiol. 82, 114 (2011).</li><li>Nyholm, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Vibrio+fischeri+and+nonsymbiotic+bacteria+in+the+dynamics+of+mucus+secretion+during+symbiont+colonization+of+the+Euprymna+scolopes+light+organ.">Roles of Vibrio fischeri and nonsymbiotic bacteria in the dynamics of mucus secretion during symbiont colonization of the Euprymna scolopes light organ.</a> Appl. Environ. Microbiol. 68, 5113 (2002).</li><li>Yip, E. S., Grublesky, B. T., Hussa, E. A., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+conserved+cluster+of+genes+promotes+symbiotic+colonization+and+Ï&#x0192;54-dependent+biofilm+formation+by+Vibrio+fischeri.">A novel, conserved cluster of genes promotes symbiotic colonization and Ï&#x0192;54-dependent biofilm formation by Vibrio fischeri.</a> Mol. Microbiol. 57, 1485 (2005).</li><li>Mandel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+regulatory+gene+is+sufficient+to+alter+bacterial+host+range.">A single regulatory gene is sufficient to alter bacterial host range.</a> Nature. 458, 215 (2009).</li><li>Graf, J., Dunlap, P. V., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+transposon-induced+motility+mutations+on+colonization+of+the+host+light+organ+by+Vibrio+fischeri.">Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.</a> J. Bacteriol. 176, 6986 (1994).</li><li>Geszvain, K., Visick, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hybrid+sensor+kinase+RscS+integrates+positive+and+negative+signals+to+modulate+biofilm+formation+in+Vibrio+fischeri.">The hybrid sensor kinase RscS integrates positive and negative signals to modulate biofilm formation in Vibrio fischeri.</a> J. Bacteriol. 190, 4437 (2008).</li><li>Krasteva, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+cholerae+VpsT+regulates+matrix+production+and+motility+by+directly+sensing+cyclic+di-GMP.">Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP.</a> Science. 327, 866 (2010).</li><li>Romling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+rdar+morphotype,+a+multicellular+behaviour+in+Enterobacteriaceae.">Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae.</a> Cell Mol. Life. Sci. 62, 1234 (2005).</li><li>Berleman, J. E., Chumley, T., Cheung, P., Kirby, 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=Rippling+is+a+predatory+behavior+in+Myxococcus+xanthus.">Rippling is a predatory behavior in Myxococcus xanthus.</a> J. Bacteriol. 188, 5888 (2006).</li><li>Lee, K., Veeranagouda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicrocells+form+by+reductive+division+in+macroscopic+Pseudomonas+aerial+structures.">Ultramicrocells form by reductive division in macroscopic Pseudomonas aerial structures.</a> Environ. Microbiol. 11, 1117 (2009).</li></ol>

Production and Free Access to this article is sponsored by Zeiss, Inc.

In this work, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model organism. Specifically, we utilize a dissecting microscope with camera attachment to monitor biofilm formation and development as wrinkled colony formation over time on a solid agar surface. In this protocol, we outline two specific types of methods we commonly use to assess wrinkled colony formation. The first is the end-point assay, which allows us to observe the final, overall 3D architecture, patterning, and diameter of a spotted culture at a selected "final" time point. This approach is most useful for assessing mutant strains or conditions that lead to dramatic defects in biofilm formation. However, this approach does not distinguish between more subtle differences occurring at time points before the selected end-point. To more closely monitor wrinkled colony formation, we use a time course assay, which allows us to identify the start of wrinkled colony formation and watch its development over time. As a result of this approach, more subtle differences in timing of wrinkled colony formation, 3D architecture, and patterning can be identified. We used this time course assay to generate two semi-quantitative assays of biofilm formation. First, the time at which a strain begins to develop 3D architecture can be compared to that of control strains. We have found that the delay in biofilm formation of a particular mutant under the same conditions is fairly consistent 9. For example, in the data shown in Fig. 2, the mutant consistently exhibited about a 4 h delay in initiating biofilm formation. A second semi-quantitative measure of biofilm formation is the change in size of the diameter of the wrinkled colony (spot). We have found that the diameter of wrinkled colonies progressively differs from that of non-biofilm colonies, reaching about a 2-fold difference at the end time point (Fig. 3) (Morris and Visick, unpublished data). To date, we have not observed one phenotype without the other (i.e., biofilm formation without an increase in colony diameter) (unpublished data), although it remains possible some mutants will behave differently. Indeed, it has been reported for V. cholerae that some wrinkled colonies result in a substantial increase in colony diameter while others do not 15. Still, assessing the change in diameter over time could aid in the characterization of potential biofilm mutants and/or provide an additional quantitative measure of mutants with delays in development. Of the two measures of biofilm formation (time and diameter), determining the start of wrinkled colony formation is more sensitive, but also more subjective, than determining the diameter of the spot. Even so, both measures provide a semi-quantitative assessment of a phenotype that is extremely useful to biofilm researchers but not readily amenable to quantification.
When performing spotted cultured assays, it is important to consider the environmental conditions in which the spotted strains are cultured. Wrinkled colony formation is often influenced by various environmental conditions, including nutrient availability, temperature, and humidity. To reduce variability between experiments, it is helpful to standardize these conditions as much possible (i.e. standardizing the agar plates to a set volume and culturing the spotted strains at a controlled temperature). To further control for variability between spotting experiments, is important to include the appropriate control strains within each set of experiments. Finally, when interpreting the data from these assays, it is necessary to perform any one experiment multiple times (3+), especially when assessing subtle differences in wrinkled colony formation (e.g., a delay in biofilm formation or patterning differences). Some limitations of this protocol are: 1) determining whether cells have a defect in growth may be difficult: growth of cells in liquid culture may not accurately reflect growth rates on solid media, and an accurate determination of growth of biofilm-forming cells, which may stick together, may not be possible; 2) strains with growth defects will be problematic to analyze; 3) it may not be possible to distinguish diameter differences between strains with subtle biofilm phenotypes; 4) for strains that do not grow in a concentric ring it may not be possible to accurately measure changes in diameter; 5) while patterning can be observed during biofilm formation, there is no way to quantify the patterning of the resulting biofilm; and 6) there is no way to measure the Z-dimension of the biofilm with this experimental set-up. Despite these limitations, this protocol nevertheless provides a means to obtain numerical data to aid in assessing wrinkled colony formation.
In this protocol, we utilize a specific imaging system (i.e., a Zeiss dissecting microscope and ProgRes CapturePro imaging software) to observe and evaluate wrinkled colony formation. The imaging system described here is powerful: the ability to detect the start of wrinkled colony formation, and thus assess development with a time course approach, is greatly enhanced through the use of a dissecting microscope. However, if this technology is unavailable, the protocol can be adapted for use with other equipment, including a simple digital camera with a zoom focus. While this protocol focuses on assessment of wrinkled colony development, it could also be modified to evaluate pellicle formation, a form of biofilm that develops when cells are growing statically in liquid culture. This protocol may also prove useful in assessing other indicators of biofilm formation, including the incorporation of specific dyes into the spotted colonies during biofilm development. These include, but are not limited to, dyes such as Congo Red and calcofluor which can bind to cellulose, a common component of bacterial biofilms 16. Additionally, this protocol, although developed for use with V. fischeri, is not limited to this organism but can be generalized to studying biofilm formation in numerous different organisms, such as Bacillus subtilis 4, Vibrio cholerae 5 , Vibrio parahaemolyticus 6, and Pseudomonas aeruginosa 7, which all exhibit wrinkled colony formation. Finally, it also can be adapted to study other colony morphologies that have a developmental pattern, such as, potentially, the patterning that occurs during growth of Myxococcus xanthus 17 and aerial structure development in Pseudomonas aeruginosa 18. This protocol is thus of general use to microbiology and biofilm researchers.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Zeiss stemi 2000-C Dissecting Microscope</td>
			<td>Zeiss</td>
			<td>45505300000000000</td>
			<td>(obtained through Lukas Microscope)</td>
		</tr>
		<tr>
			<td>ProgRes C10PLUS</td>
			<td>JENOPTIK Optical Systems GmbH</td>
			<td>017953-602-26</td>
			<td>Camera attachment (obtained through Lukas Microscope)</td>
		</tr>
		<tr>
			<td>CL 1500 EC Cold Light Source</td>
			<td>Zeiss</td>
			<td>4355400000000000</td>
			<td>(obtained through Lukas Microscope)</td>
		</tr>
		<tr>
			<td>ProgRes CapturePro</td>
			<td>JENOPTIK Optical Systems GmbH</td>
			<td>Free software with registered camera</td>
			<td>Computer program for image capture</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td>Free software from:<a href="http://rsb.info.nih.gov/ij/download.html" target="_blank"> http://rsb.info.nih.gov/ij/download.html</a></td>
			<td>Computer program for diameter measurements</td>
		</tr>
	</tbody>
</table>

a semi-quantitative approach to assess biofilm formation using wrinkled colony development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

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A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

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Nanyang Technological University

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JoVE Journal

Biology

23.2K Views.  Loyola University Medical Center. We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Video: A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots located in Xinjiang Province, northwest China. To prevent high correlations among observations from the same sampling unit, we developed the model using a linear mixed-effects approach with random plot effect to account for stochastic variability. Various tree- and stand-level variables, such as indices for tree size, competition, and site condition, were included as fixed effects to explain the residual variability. In addition, heteroscedasticity and autocorrelation were described by introducing variance functions and autocorrelation structures. The optimal linear mixed-effects model was determined by several fit statistics: Akaike&#8217;s information criterion, Bayesian information criterion, logarithm likelihood, and a likelihood ratio test. The results indicated that significant variables of individual-tree basal area increment were the inverse transformation of diameter at breast height, the basal area of trees larger than the subject tree, the number of trees per hectare, and elevation. Furthermore, errors in variance structure were most successfully modeled by the exponential function, and the autocorrelation was significantly corrected by first-order autoregressive structure (AR(1)). The performance of the linear mixed-effects model was significantly improved relative to the model using ordinary least squares regression.

Mixed-effects models are flexible and useful tools for analyzing data with a hierarchical stochastic structure in forestry and could also be used to significantly improve the performance of forest growth models. Here, a protocol is presented that synthesizes information relating to linear mixed-effects models.

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots ...