We thank Rachael B. Chanin&#160;and Alejandro Llanos-Chea for technical assistance. We thank Anthony T. Maurelli, Bryan P. Hurley, Alessio Fasano, Brett E. Swierczewski, and Bobby Cherayil for the strains used in this study. This work was supported by the National Institute of Allergy and Infectious Diseases Grant K22AI104755 (C.S.F.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Preparation of Reagents
<ol>
	<li>Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile salts in 50 mL autoclaved TSB. Filter sterilize using a 0.22 &#181;m filter. Make fresh medium weekly. 
	Notes: The bile salts routinely used is a 1:1 mixture of sodium cholate and sodium deoxycholate isolated from ovine and bovine gallbladders. As demonstrated previously4, the presence of glucose was required for bile salt-induced biofilm formation. TSB has added glucose relative to Luria-Bertani (LB) broth; and therefore, was sufficient to induce biofilm formation in Shigella and the other enteric pathogens analyzed. Depending on the bacteria to be analyzed, different glucose concentrations or different sugar requirements might be needed.</li>
	<li>0.5% w/v crystal violet in water: Dissolve 2.5 g of crystal violet in 500 mL distilled water. Filter sterilize using a 0.22 &#181;m filter.</li>
	<li>Concanavalin A (ConA) conjugated to fluorescein isothiocyanate (FITC): Reconstitute the stock in 1x PBS. Dilute the 10 mg concentrated stock with 400 &#956;L of 1x PBS to a final concentration of 25 &#181;g/mL, and protect from light.</li>
	<li>PBS + Glucose: Dissolve 0.2 g glucose in 10 mL 1x PBS (2% w/v glucose final). Filter sterilize using a 0.22 &#181;m filter. Make fresh on the day of use.</li>
	<li>PBS + Bile Salts: Dissolve 40 mg in 10 mL 1x PBS (0.4% w/v bile salts final). Filter sterilize using a 0.22 &#181;m filter. Make fresh on the day of use.</li>
	<li>PBS + glucose and bile salts: Dissolve 40 mg bile salts and 0.2 g glucose in 10 mL 1x PBS (0.4% w/v bile salts and 2% w/v glucose final). Filter sterilize using a 0.22 &#181;m filter. Make fresh on the day of use.</li>
	<li>Prepare LB agar plates.</li>
	<li>Formaldehyde/glutaraldehyde fix: Add 810 &#181;L formaldehyde (37% stock solution, 3% final concentration) and 125 &#181;L glutaraldehyde (25% stock solution, 0.25% final concentration) to 14 mL 1x PBS. Mix thoroughly and store at 4 &#176;C. The fix should be cold for proper use. 
	Caution: The fix is toxic and requires hazardous waste disposal.</li>
	<li>Antifade mountant solution: Use antifade mountant solution containing 4,6-diamidino-2-phenylindole (DAPI) stain to inhibit photobleaching of immunofluorescent microscopy samples while fluorescently staining the DNA of the bacteria.</li>
</ol>
2. Preparation of Bacteria
<ol>
	<li>Grow overnight cultures of the bacterial strains to be tested by inoculating 3 mL of TSB with a single, well-isolated colony in a sterile culture tube. Incubate at 37 &#176;C with shaking at 225 rpm for overnight incubation (16 - 24 h). 
	NOTE: Strains should be restreaked from freezer stocks every 2 to 4 weeks, and maintained on plates no more than 2 weeks old.</li>
</ol>
3. Solid-phase Adherence Assay
NOTE: This assay quantifies adherent bacteria using a 96-well plate method. Bacteria are grown statically in flat bottom plates. Washing is performed to remove non-adherent bacteria and adherent bacteria are stained with crystal violet. The crystal violet stain binds peptidoglycan in the bacterial cell wall and can be solubilized using ethanol. The number of adherent bacteria is determined based on crystal violet retention.
<ol>
	<li>Set up two 1.5 mL tubes. Label with TSB or TSB + bile salts (BS).</li>
	<li>Add 1 mL of TSB or TSB + BS to the respective tubes.</li>
	<li>Inoculate tubes with 20 &#181;L of overnight culture (at a 1:50 dilution).</li>
	<li>In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 &#181;L/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type (TSB and TSB + BS) to be tested.</li>
	<li>Add 130 &#181;L/well of inoculated culture into three wells, and repeat until all experimental conditions are plated in triplicate.</li>
	<li>Incubate for 4 - 24 h at 37 &#176;C statically.</li>
	<li>Using a plate reader, record the OD600. Set the control wells as &#39;blank.&#39;&#160;Confirm the control medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD600 values can be used to normalize the data if there are significant differences in growth rate between bacterial strains.</li>
	<li>Remove the culture medium using a vacuum line by gently tilting the plate and slowly aspirating the medium at the lower edge of the well. Be sure to collect all the culture medium without disrupting the adherent bacterial population located on the plastic surface. If EPS matrix was produced during the incubation time, the matrix will be visualized as a white precipitate. Do not disturb the EPS matrix.</li>
	<li>Gently wash the wells once with 200 &#181;L sterile PBS. Remove the PBS wash using the vacuum line.</li>
	<li>Invert the plate to dry. Allow a minimum of 20 min to dry. 
	NOTE: Since the biofilm must be thoroughly dried before staining, the protocol can be paused at this step for a few hours or even overnight. The added drying time will not alter the staining procedure while incomplete drying will affect quantification of the results and reproducibility.</li>
	<li>Add 150 &#181;L of 0.5% crystal violet to each experimental and control well.</li>
	<li>Incubate for 5 min at room temperature (RT).</li>
	<li>Wash the wells once with 400 &#181;L of distilled water. The added volume helps to remove residual crystal violet stain from the sides of the wells. Remove the wash with the vacuum line.</li>
	<li>Wash the wells five times with 200 &#181;L of distilled water. Remove the wash with the vacuum line. 
	NOTE: Thorough washing is important for quantification. When the blank wells are clear from the distilled water and do not contain any residual crystal violet stain, proceed to the next step.</li>
	<li>Invert the plate to dry, protected from light. Ensure complete drying of the plate as noted above.</li>
	<li>Destain the wells with 200 &#181;L of 95% ethanol. Incubate the plate on the shaker for 30 min. To avoid evaporation, particularly at higher temperatures, perform this step at 4 &#176;C.</li>
	<li>Using a plate reader, record the OD540. 
	NOTE: The wavelength for maximum absorbance of crystal violet is near 590 nm. The literature reports a range from OD540&#160;- OD5954,13,14,15,16 for crystal violet absorbance; thus choose a wavelength available based on the plate reader.</li>
</ol>
4. EPS Matrix Detection
NOTE: These complimentary assays quantify and visualize the EPS. In both, EPS is detected using a lectin to bind polysaccharides. The fluorescently-conjugated protein allows quantification (step 4.1) or visualization (step 4.2).
<ol>
	<li>Semi-quantitative detection of EPS
	<ol>
		<li>Set up two 1.5 mL tubes. Label with TSB or TSB + BS.</li>
		<li>Add 1 mL of TSB or TSB + BS to the respective tubes.</li>
		<li>Inoculate tubes with 20 &#181;L of overnight culture (a 1:50 dilution).</li>
		<li>In a sterile, black, flat-bottomed, tissue culture-treated 96-well plate, add 130 &#181;L/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each medium type to be tested. Add 130 &#181;L/well of inoculated culture to the wells, and repeat until all experimental conditions are plated in triplicate.</li>
		<li>Incubate for 4 - 24 h at 37 &#176;C statically.</li>
		<li>Transfer the culture medium to a clear 96-well plate with a pipette, ideally a multichannel pipette. Be cautious to collect all the culture medium without disrupting the adherent population and/or the EPS located on the plastic surface. Set aside.</li>
		<li>Fix the black plate for 15 min at RT using 200 &#181;L/well of the formaldehyde/glutaraldehyde in 1x PBS.</li>
		<li>While the adherent population is fixing, evaluate the supernatant fraction from step 4.1.6. Using a plate reader, record the OD600. Set the control wells as &#39;blank.&#39;&#160;Confirm the control medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment.
		<ol>
			<li>Alternatively, the entire assay can be performed in a black clear bottom plate. In those circumstances, the OD600 values can be recorded and the culture medium can be subsequently discarded prior to proceeding to step 4.1.7. The OD600 values can be used to normalize the data in the case there are significant differences in growth rate between bacterial strains.</li>
		</ol>
		</li>
		<li>Remove the fix and dispose in the hazardous waste.</li>
		<li>Gently wash the wells twice with 200 &#181;L/well of sterile PBS. Remove the PBS wash using the vacuum line by gently tilting the plate and slowly aspirating the wash at the lower edge of the well.</li>
		<li>Add 150 &#181;L/well of 25 &#181;g/mL ConA-FITC, and incubate for 15 min at RT.</li>
		<li>Gently wash twice with 200 &#181;L of PBS.</li>
		<li>Add 150 &#181;L of PBS to each well. Record the fluorescence at 488 nm.</li>
	</ol>
	</li>
	<li>Confocal microscopy visualization of EPS production and calculation of biofilm thickness
	<ol>
		<li>Set up two 1.5 mL tubes. Label with TSB or TSB + BS.</li>
		<li>Add 1 mL of TSB or TSB + BS to the respective tubes.</li>
		<li>Inoculate tubes with 20 &#181;L of overnight culture (at a 1:50 dilution).</li>
		<li>Set up a sterile 24-well plate with 12 mm sterile round glass coverslips. Add 400 &#181;L/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each medium type to be tested.</li>
		<li>Add 400 &#181;L of inoculated culture to wells in duplicate, and repeat until all experimental conditions are plated.</li>
		<li>Incubate for 4 - 24 h at 37 &#176;C statically.</li>
		<li>Visually confirm growth in the TSB condition, growth and EPS precipitate (white) in the TSB + BS condition, and no growth and/or white precipitate in sterile control wells. Remove the supernatants.</li>
		<li>Fix for 15 min at RT using 200 &#181;L/well of formaldehyde/glutaraldehyde solution in 1x PBS.</li>
		<li>Remove the fix and dispose in hazardous waste.</li>
		<li>Gently wash the wells twice with 200 &#181;L/well of sterile PBS. Remove the PBS wash using the vacuum line.</li>
		<li>Add 150 &#181;L/well of 25 &#181;g/mL ConA-FITC and incubate for 15 min at RT.</li>
		<li>Gently wash twice with 200 &#181;L/well of PBS.</li>
		<li>Mount with antifade mountant solution with DAPI. 
		NOTE: The solution is a ready-made, glycerol-based mount solution containing DAPI for preserving the fluorescent label while simultaneously staining the DNA in bacterial cells for visualization as a counterstain. Follow the kit&#39;s directions and recommendations for incubation times prior to imaging samples. The DAPI staining procedure can be performed prior to applying antifade mountant solution that does not contain DAPI.</li>
		<li>Evaluate by confocal microscopy. On a confocal microscope, set the laser to 495 nm/519 nm excitation/emission for FITC visualization. Set a second laser to 360 nm/460 nm excitation/emission to visualize DAPI. Locate the biofilm by focusing on the DAPI channel. Use both the DAPI and FITC channels to determine the full thickness and set the upper and lower imaging borders. Record full thickness Z-stack images by capturing images every 0.25 &#181;m after setting perimeters to the top and bottom of the z-stack. For more information on confocal microscopy, please refer to publications by Paddock et al.17,18,19,20</li>
		<li>Reconstruct the 3D images in ImageJ (https://imagej.nih.gov/ij/) to visualize the full biofilm formation and calculate the biofilm thickness. The average thickness obtained for S. flexneri bile-salt induced biofilms was 14 &#181;m4.</li>
	</ol>
	</li>
</ol>
5. Dispersion Assay
NOTE: In this assay, the disassembly of biofilm through bacterial dispersion is detected. Here, mature biofilms are established and subsequently (usually the next day), the media are replaced with PBS or supplemented PBS. The supernatant component is then assessed to quantitate the number of bacteria that have dissociated from the biofilm.
<ol>
	<li>Set up two 1.5 mL tubes. Label with TSB or TSB + BS.</li>
	<li>Add 1 mL of TSB or TSB + BS to the respective tubes.</li>
	<li>Inoculate tubes with 20 &#181;L of overnight culture (at a 1:50 dilution).</li>
	<li>In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 &#181;L/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type to be tested.</li>
	<li>Add 130 &#181;L/well of inoculated culture to three wells, and repeat until all experimental conditions are plated in triplicate.</li>
	<li>Incubate the plate for 4 - 24 h at 37 &#176;C statically.</li>
	<li>Before continuing, warm the PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose to 37 &#176;C. Prepare these reagents fresh daily.</li>
	<li>Using a plate reader, record the OD600. Set the control wells as &#39;blank.&#39;&#160;Confirm the medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD600 values can be used to normalize the data if there are significant differences in growth rate between bacterial strains.</li>
	<li>Remove the culture medium using a vacuum line. Be sure to collect all the culture medium without disrupting the adherent population located on the plastic surface.</li>
	<li>Gently wash the wells twice with 200 &#181;L/well of sterile PBS. Remove the PBS wash using the vacuum line.</li>
	<li>Replace the wash with 130 &#181;L/well of the following into three wells each: PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose. These reagents must be pre-warmed to 37 &#176;C.</li>
	<li>Incubate the plate for 30 min at 37 &#176;C.</li>
	<li>Carefully remove the plate from the 37 &#176;C incubator. Transfer the supernatants to a fresh, sterile 96-well plate.</li>
	<li>Using a 96-well plate or dilution block, prepare 10-fold (1:10) serial dilutions of the supernatant into sterile PBS. 
	NOTE: The dilution series should range from undiluted to 10-6 depending on the bacterial strain to be tested. Pilot experiments can be performed to determine the optimal dilution range.</li>
	<li>Spot plate 5 &#181;L of each dilution onto LB agar using a multichannel pipette. Incubate at 37 &#176;C overnight. Count colonies on the next day and account for the dilution factor when determining the recovery colony forming units (CFU). To calculate the percent dispersion relative to the 1x PBS control (set at 100%), divide the recovery CFU from each treatment condition by the recovery CFU from the 1x PBS control sample. 
	NOTE: Routine media plates for the bacterial strain of interest can also be used. For example, Shigella can be plated on Congo red plates. Ensure the plates are dry for appropriate spot-plating techniques.</li>
</ol>

The authors have no disclosures.

Analysis of biofilm formation is challenging due to the dynamic nature of biofilms and the variability between strains, materials, laboratories, and assays. Here, several strategies are presented to determine biofilm formation in enteric pathogens following bile salts exposure with experimental insight provided to promote reproducibility. There are additional considerations to ensure reproducibility. First and foremost, we recommend performing at least three independent experiments each with technical triplicates to conf...

Biofilm formation is an important bacterial survival strategy induced during harsh environmental conditions. Exposure to bactericidal compounds like antibiotics or changes in nutrient or oxygen availability induces a stressed state in bacteria that can be alleviated through biofilm formation. A biofilm is characterized by bacterial attachment to a surface or other bacteria and is accompanied by the secretion of an EPS matrix primarily composed of polysaccharides1,2,3. Biofilm formation is a dynamic process in which a cascade of events culminates in formation of a mature adherent bacterial community1,2,3. Bacteria produce adhesins to facilitate early attachment while shifting adhesin gene expression profiles to strengthen attachment during biofilm maturation. Simultaneously, EPS production occurs to coat the bacterial community in a matrix to protect the cells from the initial stressor. Bacteria contained within the biofilm are slow growing; and as such, renders most antibiotics ineffective. Furthermore, the slow growth conserves energy until conditions change to favor bacterial growth1,2,3. After the harsh conditions have passed, bacteria disperse the biofilm and resume a planktonic lifestyle1,2,3. Traditionally, biofilms are observed on surfaces and represent a persistent clinical challenge due to infection reservoirs present on catheters and in-dwelling devices1,2,3.
Biofilm formation was recently described for several enteric pathogens; bacteria that infect the small intestine or colon4. For Shigella species, infection occurs in the human colon after a transit through the majority of the gastrointestinal tract. During passage through the small intestine, Shigella is exposed to bile; a lipid-degrading detergent secreted into the intestine to facilitate digestion of lipids while simultaneously killing most bacteria5. Enteric pathogens have a unique ability to resist the bactericidal effects of bile6. Our recent analysis utilized in vivo-like combinations of glucose and bile salts to demonstrate robust biofilm formation in S. flexneri as well as other species of Shigella, pathogenic Escherichia coli, and Salmonella4. Previously, Salmonella enterica serovar Typhi was shown to form a bile-induced biofilm due to unique colonization of the gallbladder during chronic infection7,8,9,10. Additionally, prior research with Vibrio11&#160;and Campylobacter12 demonstrated biofilm formation in response to bile. Therefore, the analyses extended the bile-induced biofilm formation observations to other pathogens and help to establish demonstration of a conserved enteric pathogen response to bile. Unlike chronic biofilms in which bacterial gene transcription is limited and cell senescence can occur1,2,3, we propose that the enteric bile-induced biofilm is more transient in nature. This transient, virulent biofilm is hallmarked by a rapid disassembly (as seen in the dispersion assay) and enhanced virulence gene expression observed in the biofilm population4,6.
As biofilm formation is a multifaceted, dynamic process and the use of bile salts as an initiating factor has only been recently described for most enteric pathogens, the tools and techniques used are unique and creative applications of traditional methods. Thus, presented here are three complimentary strategies to quantify several important characteristics of bile salt-induced biofilm formation, including bacterial adherence, production of the EPS matrix, and dispersion of viable bacteria from the biofilm. These techniques have been utilized primarily for research with Shigella; and therefore, evaluation of other enteric pathogens may require optimization. Nevertheless, positive data from all three assays support identification of biofilms and establish reproducible protocols for bile salt-induced biofilm formation.

<table>
	<tbody>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma</td>
			<td>C6158-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin-A FITC</td>
			<td>Sigma</td>
			<td>C7642-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bile Salts</td>
			<td>Sigma</td>
			<td>B8756-100G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma</td>
			<td>L7533-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL culture tubes, 17 x 100 mm, plastic, sterile</td>
			<td>Fisher</td>
			<td>14-959-11B</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield hard-set antifade with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F1635-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 96-well plates (clear)</td>
			<td>TPP</td>
			<td>92696</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 96-well plates (black)</td>
			<td>Greiner Bio-One&#160;</td>
			<td>655076</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 24-well plates (clear)</td>
			<td>TPP</td>
			<td>92424</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 12mm, round</td>
			<td>Fisher</td>
			<td>08-774-383</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate reader</td>
			<td>Spectramax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flourescent plate reader</td>
			<td>Biotek Synergy 2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal or Fluorescent Microscope</td>
			<td>Nikon A1 confocal microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Shaking Incubator</td>
			<td>New Brunswick Scientific Excella E25</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Plate Incubator</td>
			<td>Thermolyne Series 5000</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bile salt-induced biofilm formation in enteric pathogens: techniques for identification and quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

In Figure 1, biofilm formation is induced in most of the six enteric pathogens tested following growth in media containing bile salts. A significant increase in adherent bacteria after bile salts exposure is observed in nearly all strains tested. The exception is enteroaggregative E. coli (EAEC); however, note the induced observation of the &#916;aaf mutant4. The results indicate that additional adherence mec...

Watch this Scientific Journal Video about Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification at JoVE.com

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

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Immunology and Infection

12.3K Views.  Massachusetts General Hospital. This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Video: Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

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 ...

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. ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

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. ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...