Electron microscope imaging was conducted at the Electron Microscopy Unit of the Weizmann Institute of Science, supported in part by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. This research was also supported by the ISF I-CORE grant 152/1, Mr. and Mrs. Dan Kane, Ms. Lois Rosen, by a Yeda-Sela research grant, by the Larson Charitable Foundation, by Ruth and Herman Albert Scholars Program for New Scientists, by the Ilse Katz Institute for Materials Sciences and Magnetic Resonance Research grant, by the Ministry of Health grant for alternative research methods, and by the France-Israel Cooperation - Maimonide-Israel Research Program. IKG is a recipient of the Rowland and Sylvia Career Development Chair.

1. Assessing the Effect of Small Molecule Inhibitors on Pellicle and Biofilm Colony Formation
<ol>
	<li>Prepare a 2x solution of defined biofilm-inducing MSgg medium25 without the calcium chloride and iron(III) chloride hexahydrate. After filter sterilization, add the calcium chloride. The medium is ready to use directly or it can be stored at 4 &#176;C in the dark.</li>
	<li>Prepare the 1x MSgg dilution on the day of the experiment.
	<ol>
		<li>Dilute the 2x MSgg medium to 1x with sterile distilled water (pellicles) or sterile 3% hot (80 &#176;C) agar (biofilms) and add iron(III) chloride hexahydrate to a final concentration of 50 &#956;M (pellicles) or 250 &#181;M (biofilms). Add antibiotics or small molecule inhibitors to the desired concentration and mix well. For example, to obtain a final concentration of 0.5 mM D-leucine in 30 ml to establish pellicles or biofilms, add 196.6 &#181;l of a 76.3 mM (10 mg/ml) D-leucine stock solution. 
		NOTE: The final 1x MSgg composition is described in Table 1. Compared to the original recipe25, the medium contained 50 &#181;g/ml threonine and the iron concentration to grow biofilm colonies on solid MSgg medium was increased 2.5x to optimize the wrinkled colony morphology.</li>
	</ol>
	</li>
	<li>After solidification of the agar, dry the solid MSgg plates in a biological hood for 30-45 min prior to the inoculation.</li>
	<li>To select specific inhibitors that interfere with the mechanisms of pellicle formation (Figure 1), rule out that the concentrations used affect planktonic and static growth.
	<ol>
		<li>Determine planktonic growth (increase in optical density over time in liquid culture) in a simple growth curve by measuring the optical density at 600 nm every hour until the stationary growth phase.</li>
		<li>To confirm that the measured culture turbidity represents live cell counts, determine the numbers of colony forming units (CFU) of cells in the planktonic growth phase from a shaking culture after several time points.</li>
		<li>To assess the effect of small molecule inhibitors on static pellicle growth, harvest cells at the end of a 3-day incubation at 23 &#176;C from a 24-well cell-culture well, inoculated under the same conditions as described in sections 1.7-1.9 and determine the CFU. For this control, use a pellicle-deficient strain that lacks the operons encoding for the extracellular matrix components (i.e., B. subtilis &#916;epsH, &#916;tasA). 
		NOTE: This strain is capable of growing under static conditions, but in contrast to a pellicle-forming wild type, it is deficient in the ability to float to the liquid-air interface, where growth is favored due to increased oxygen levels26. Thus, this extracellular matrix- and pellicle-deficient strain is a recommended reference strain to assess growth under static conditions. 
		NOTE: For the specific example of the non-canonical D-amino acid D-leucine described below, an effect on planktonic and static growth at concentrations that interfered with pellicle formation was ruled out12,17. The methods to determine planktonic and static growth are described in detail17.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54612/54612fig1.jpg" /> 
Figure 1. Conceptual overview for the identification of a robust experimental setup to assess the specific inhibition of biofilm formation. Selection criteria for small molecule inhibitors that indicate specific interference with biofilm formation without pronounced effect on planktonic growth. <a href="https://www.jove.com/files/ftp_upload/54612/54612fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Streak out B. subtilis from a -80 &#176;C stock (LB culture of 109 cells/ml frozen in 20% glycerol) to isolate single colonies on a LB-1.5% agar plate with a sterile tip or applicator stick.</li>
	<li>Grow overnight at 30 &#176;C.</li>
	<li>CRITICAL STEP: For a robust pellicle inhibition by the non-canonical D-amino acids such as D-leucine, grow a single colony picked from the LB-1.5% agar plate in 3 ml LB broth at 37 &#176;C for 4 hr in a shaking incubator (shaking speed 200 rpm). Replace the LB broth with biofilm-inducing MSgg medium prior to inoculation by centrifuging 1.5 ml starter culture for 4 min at 6,000 x g, carefully removing the supernatant and re-suspending the pellet in 1.5 ml MSgg medium. The rest of the culture can be discarded. 
	Important: To ensure the robustness of the system, the optical density at 600 nm (OD600) of the washed starter culture should be between 0.6 and 1.</li>
	<li>During the growth of the starter culture, prepare a 12-well cell-culture multidish plate containing 3 ml of MSgg medium without or with a concentration range of the small molecule inhibitor (e.g., 0.3, 0.5, 1 mM D-leucine17). To rule out edge effects, distribute the location of the different concentrations across the multidish plate. Alternatively, use 24-well cell-culture multidish plates containing 1.5 ml of MSgg medium.</li>
	<li>Inoculate the wells of the 12-well cell-culture multidish plate with 3 &#181;l of the washed starter culture (1:1,000 dilution). 
	NOTE: A lower dilution ratio, i.e., 1:500 can be used. This decreases the development time of the pellicles.</li>
	<li>Grow the pellicles at 23 &#176;C under static conditions for three days. Do not move the pellicles during this time, as it can affect the final surface morphology of the pellicle.</li>
	<li>Acquire pictures with a binocular and homogenous exposure of lightning. Alternatively, take a picture of the pellicles with a high resolution camera. To avoid artefacts caused by inconsistent light angles and shadows, take top-down pictures with the camera fixed on a tripod and use a soft and large light source at 45&#176; from both sides. 
	NOTE: An alternative method to study B. subtilis multicellularity is the biofilm colony assay on solid, biofilm-inducing MSgg medium. Like pellicles, this assay allows the study of spatiotemporal processes. Once the active range of small molecule inhibitors is determined, their effect on biofilm colony formation can be studied.</li>
	<li>To grow biofilm colonies, symmetrically spot 1.5 &#181;l of the unwashed pre-culture (Step 1.7) on the dried MSgg 1.5% agar plate with the help of a template &#8212; 4 drops per Petri dish of 8.5 cm diameter. Let the drops adsorb to the plate before moving them. 
	NOTE: The template helps to get an equal distribution of the biofilm colonies within the area where the cells are grown. To prepare the template, draw the total area of growth at original scales, divide it to equal sectors and mark the center. For a round Petri dish of 8.5 cm diameter, this assigns 14 cm2 to one biofilm colony.</li>
	<li>Incubate the plates at 30 &#176;C for three days. During this time, biofilm colonies develop and form a three-dimensional, wrinkled structure.</li>
	<li>Take pictures as in step 1.11.</li>
</ol>
2. Ethanol Resistance Assay
<ol>
	<li>Grow biofilms as described in steps 1.1-1.7 and 1.12-1.14.</li>
	<li>After 68 hr of growth at 30 &#176;C, cut the biofilm colonies into two equal parts with the help of a razor blade and the template.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54612/54612fig2.jpg" /> 
Figure 2. Example experimental design to assess the resistance of biofilm colony cells to sterilizing agents. (A) Template used for the equal distribution of biofilm colonies across a Petri dish and for cutting. (B) Top-down images of untreated wild-type biofilm grown for 68 hr on solid, defined biofilm-inducing MSgg medium at 30 &#176;C. The enlargement shows how a biofilm colony can be cut in two equal halves. (C) The two equal biofilm halves are treated equally (control, PBS) or with either PBS or sterilizing agent and processed as described. Scale bar: 1 cm. <a href="https://www.jove.com/files/ftp_upload/54612/54612fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Carefully lift each half of the biofilm colony from the agar plate with a small spatula and move it to a 1.5 ml microcentrifuge tube containing 500 &#181;l of phosphate-buffered saline (PBS). If necessary, scrape the remaining cells from the plate and transfer them to the microcentrifuge tube as well. 
	NOTE: The second half of the biofilm colony is treated differentially, depending on whether it is the control or to test the resistance to sterilizing agents.</li>
	<li>For the control, incubate the second half of the biofilm colony in 500 &#181;l PBS as in step 2.3. To assess resistance to sterilizing agents, transfer the second half of the biofilm colony to 500 &#181;l 50% (v/v) ethanol. 
	NOTE: Alternative sterilizing agents such as sodium hypochlorite can be used. For all sterilizing agents used, determine the active concentration and incubation time in a preliminary experiment.</li>
	<li>Incubate the biofilm colonies for 10 min on the bench-top at room temperature.</li>
	<li>Centrifuge the biofilm colonies for 5 min at 18,000 x g and carefully remove the supernatant with a pipette. Add 300 &#181;l of PBS.</li>
	<li>Sonicate the cells mildly (amplitude 10%, pulse 5 sec) with the microtip of a sonicator. 
	NOTE: The sonication energy must be sufficient to separate biofilm aggregates. However, too harsh sonication may lyse the cells. Confirm in advance by light microscopy that the sonication energy used does not lyse the cells and that all aggregates are dissolved.</li>
	<li>Add 700 &#181;l of PBS to a final volume of 1 ml. Perform a serial dilution (to 10-7) in PBS and spread 100 &#181;l of 3 dilutions on a LB-1.5% agar plate using sterile glass beads. 
	NOTE: The optimal dilutions to be plated should be determined in a preliminary experiment, as this depends on the amount of cells in the biofilm colony of interest and the survival rate of the cells in response to the sterilizing agent.</li>
	<li>Incubate the plates overnight at 30 &#176;C, count the CFU and determine CFU/ml. From the final CFU/ml of each the half biofilm colonies, calculate the percentage of survivors. 
	NOTE: When performed and analyzed as described, the two halves of the control biofilm colony and the untreated versus ethanol-treated half of the untreated biofilm colony should yield differences below 10% in viable cell counts, verifying the symmetry or the resistance of the colony, respectively. Alternatively, the results can be represented in total CFU. The cell counts of the control and untreated biofilm colony should remain in the same order of magnitude. In contrast, the cell counts of the ethanol-treated half of a small molecule-treated biofilm colony are expected to drop by a minimum of two orders of magnitude to claim for an increased sensitivity to the sterilizing agent.</li>
</ol>
3. Biofilm Colony Sample Preparation for Scanning Electron Microscopy
<ol>
	<li>Grow biofilm colonies as described in steps 1.1-1.7 and 1.12-1.14.</li>
	<li>Prepare a fresh batch of 2% (v/v) glutaraldehyde, 3% (v/v) paraformaldehyde solution in 100 mM sodium cacodylate, 5 mM calcium chloride buffer, pH 7.3. Prepare 5 ml of fixative for every Petri dish of 8.5 cm diameter. 
	CAUTION: Glutaraldehyde and paraformaldehyde are hazardous. Handle them with safety equipment inside a chemical hood. Discard the solutions and the contaminated materials to the hazardous waste.</li>
	<li>Carefully add the fixative to the biofilm colonies, without dispensing directly on top of the biofilms. 
	NOTE: Due the hydrophobic character of the biofilm colony, the colonies slowly detach from the agar and start to float.</li>
	<li>Carefully seal the plates with a strip of Parafilm. Incubate on a rotary shaker for 2 hr at room temperature and subsequently transfer the plates to 4 &#176;C for overnight.</li>
	<li>The next day, carefully remove the liquid with a Pasteur glass pipette connected to a vacuum pump.</li>
	<li>Carefully add 10 ml 100 mM sodium cacodylate, 5 mM calcium chloride buffer to wash the biofilm and incubate for 5 min. Gently remove the liquid with the Pasteur glass pipette from the corner of the plate to avoid damaging the biofilm and add fresh washing solution by gentle pipetting. Repeat this step once.</li>
	<li>For the dehydration of the biofilm colonies, proceed with the following steps: 2x 5 min in ddH2O; 2x 20 min in 30% ethanol; 2x 20 min in 50% ethanol; 2x 20 min in 70% ethanol; 2x 20 min in 96% ethanol; 2x 30 min in 100% ethanol.
	<ol>
		<li>Add 15 ml of liquid for every Petri dish of 8.5 cm diameter in each step and remove the liquid carefully after each incubation.</li>
	</ol>
	</li>
	<li>Use one of two different methods for drying the samples from ethanol.
	<ol>
		<li>For air-drying from ethanol:
		<ol>
			<li>Cut a cellulose filter paper (diameter of 9 cm) in quarters. Briefly submerge one quarter in 100% ethanol, and then carefully transfer one floating biofilm colony onto it. Put the wet filter paper in a Petri dish lined with a filter paper. Cover the Petri dish and let the biofilm colonies dry overnight in a chemical hood.</li>
		</ol>
		</li>
		<li>For critical point (CP)-drying using carbon dioxide (CO2) as the transition fluid:
		<ol>
			<li>Fill 75% of the critical point drying machine chamber with 100% ethanol. Transfer the samples into a holder, each sample into its own chamber. If necessary, cut the biofilm with scissors into smaller pieces. Leave the samples submerged in ethanol during all the handling. Then, transfer the holder into the chamber and close the chamber tightly.</li>
			<li>Cool the chamber to 7 &#176;C and start stirring. Fill the chamber completely with liquid CO2. During a 7 min incubation time, let the ethanol mix with the CO2. Then, discharge 25% of the solution. 
			NOTE: Do not empty the chamber below the level of the sample.</li>
			<li>Repeat step 3.8.2.2 four times.</li>
			<li>Repeat step 3.8.2.2 five times with an incubation time of 5 min only. Finally, the ethanol should be completely replaced by CO2.</li>
			<li>During the last round, empty only 5% of the chamber. Turn off the stirring and the cooling. Start heating the chamber to 42 &#176;C. At a temperature of 31.1 &#176;C and a pressure of 73.9 bar, the liquid CO2 reaches its critical point, the state where the gaseous phase has the same density as the liquid phase of the solvent27. Once the temperature reaches 42 &#176;C, incubate for 10 min. At 42 &#176;C, the CO2 in the chamber exists as supercritical gas. 
			NOTE: Constantly check the pressure of the chamber. The pressure should not exceed 120 bar at 42 &#176;C.</li>
			<li>Start to slowly release the gas with continuous heating. This keeps the samples in the CO2-gas phase and prevents the deformation of the sample morphology through the liquid surface tension. Set the flowmeter to 5 L/hr by fine tuning the metering valve controlling the flowmeter. Wait until the all the pressure in the chamber is released. Now open the chamber and remove the samples carefully from the holder.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Coat an electron microscopy stub with carbon tape. With the help of tweezers, carefully transfer the biofilm colonies onto the stub. Connect each colony to the stub by adding a thin bridge from carbon tape, which is crucial for charge elimination under the electron beam. At this stage, handle the biofilm colonies with care as they are very fragile. Store the samples in a desiccator for at least 24 hr or until examination.</li>
	<li>The day of the examination with the scanning electron microscope, sputter-coat the biofilm colonies for 2 min in a 60&#176; angle in a gold-palladium sputter-coater. Repeat this step twice and rotate the samples by 120&#176; in between. At the end, sputter-coat the samples once for 3 min from the top. The 20 nm thin layer of gold-palladium improves the conductivity and enhances the contrast of the sample for the imaging in the SEM.</li>
	<li>Store the samples in a desiccator to avoid the rehydration of the sample28 until imaging with a scanning electron microscope29,30.</li>
</ol>

The authors have nothing to disclose.

Bacillus subtilis forms robust and highly structured biofilms both in liquid (pellicles) and on solid medium (colonies). Hence, it serves as an ideal model organism to characterize the mode of action of specific biofilm inhibitors. On solid media, cells form multicellular structures with distinctive features that are not evident in pellicles, like wrinkles radiating from the center to the edge. Thus, pellicles and colonies are complementary systems to study B. subtilis multicellularity.
<p class="jo...

Multi-cellular bacterial communities play significant roles in natural and anthropogenic environments, and can be beneficial or highly deleterious. These multi-cellular colonies are known as biofilms, wherein the individual cells are embedded in a self-produced extracellular polymeric substances (EPS) matrix. The EPS strongly adhere the cells to the surface they colonize. They serve as a shield against mechanical and chemical forces and create close contact between neighboring cells, facilitating cellular communication1. A biofilm can be viewed as a differentiated community, where cells use highly regulated, orchestrated processes to coordinate their activities within the community, as well as across species2-5. The transition from a planktonic, free-living mode of growth to a biofilm state is often associated with developmental processes. A good example is the Gram-positive soil bacterium Bacillus subtilis, and therefore an undomesticated strain serves as a robust model organism to study the developmental stages leading to biofilm formation. In this bacterium, motile cells organize themselves into conspicuous multicellular structures that carry out specialized tasks4. One group of cells, the matrix-producers secrete exopolysaccharides6, the amyloid protein TasA7,8, and the surface hydrophobicity protein BslA9,10; all of which participate in the assembly of the EPS11-13.
Given the abundance of biofilms in natural and anthropogenic niches and the putative fatal damage they can cause, there is a pressing need to find ways to prevent their formation. Small molecule inhibitors can aid in the discovery of new regulatory pathways, enzymes and structural proteins involved in biofilm formation, and thereby promote insights in the complex processes of multicellular community assembly. As B. subtilis is a well-studied model for biofilm formation14,15, it can be used to assess the effects of various biofilm inhibitors. This study tackles four fundamental methods that are key for evaluating the response of biofilms to small molecule inhibitors. First, to ensure that these inhibitors have a biofilm-specific target, the separation of the effect on planktonic growth from the effect on biofilm formation is critical. Most antibacterial agents target cells in their planktonic growth phase, but molecules that target the biofilm lifestyle are rare. Additionally, as molecules that do not affect planktonic growth are not toxic, they can reduce the selective pressure favoring antibiotic resistant mutants16. For example, when biofilms are treated with D-amino acids or certain other cell wall-interfering molecules, they are either disturbed or disassembled, but these inhibitors only mildly affect planktonic growth12,17. In contrast, many antibiotics dramatically impair planktonic growth, with little or no effect on biofilm formation17.
Second, establishing a consistent and robust experimental framework to study the effect of the small molecules is crucial. We observed that the active concentration range of small molecule inhibitors was sensitive to the pre-culture conditions and to the experimental setup used to study the effect of these small molecule inhibitors. Various reports, particularly those studying B. subtilis, revealed variations in the concentration range at which D-amino acids inhibit the formation of pellicles &#8212; the floating bacterial biofilms12,17-19. The results presented here suggest that the following factors account for differences in the active concentration range: the pre-culture conditions (logarithmic12,17 versus late-stationary20 growth phase), the growth medium used in the pre-culture condition (rich, undefined [Luria Broth, LB] versus defined [monosodium glutamate-glycerol, MSgg]), the inoculation ratio and especially the removal of the pre-culture medium before inoculation. The temperature of static pellicle growth showed a less important role in the activity range of the small molecule inhibitor D-leucine, a representative D-amino acid used in this study.
Finally, once the biofilms are treated with specific biofilm inhibitors, robust and informative methods are required to characterize the effects of these inhibitors on biofilm fitness. Here, two methods to independently characterize the effect of small molecule inhibitors are described in detail: (1) The effect on single cells within a biofilm colony and their resistance to antimicrobial agents. Cells in biofilms are typically more resistant to antibiotics when compared to free-living bacteria21-23. While this phenomenon is multifactorial, the ability of the EPS to reduce antibiotic penetration was often considered as an appealing explanation24. This method assesses the survival of pre-established biofilm cells after exposure to antibacterial substances. (2) The effect on the biofilm colony architecture, from the large to the small scale. Biofilm colonies are characterized by their three-dimensional structure and the presence of the EPS. Utilizing scanning electron microscopy, changes in the cell morphology, biofilm colony structure and the architecture and abundance of the EPS can be visualized from the large (mm) to the small scale (&#181;m).

<table border="0" cellpadding="0" cellspacing="0" width="897">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Luria Broth, Lennox</td>
			<td style="width:305px;">Difco</td>
			<td style="width:127px;">240230</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate monobasic&#160;</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate dibasic&#160;</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher Scientific, 209.27 g/mol</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnesium chloride hexahydrate&#160;</td>
			<td>Merck, 203.30 g/mol&#160;</td>
			<td>1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride anhydrous</td>
			<td>J.T. Baker, 110.98 g/mol</td>
			<td>1311-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">manganese(II) chloride tetrahydrate</td>
			<td>Sigma, 197.91 g/mol</td>
			<td>31422-250G-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iron(III) chloride hexahydrate&#160;</td>
			<td>Sigma, 270.30 g/mo)</td>
			<td>F2877-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zinc chloride anhydrous&#160;</td>
			<td>Acros Organics, 136.29 g/mol</td>
			<td>424592500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thiamine hydrochloride</td>
			<td>Sigma, 337.27 g/mol</td>
			<td>T1270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-tryptophan</td>
			<td>Fisher Scientific, 204.1 g/mol</td>
			<td>BP395-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-phenylalanine</td>
			<td>Sigma, 165.19 g/mol</td>
			<td>P5482-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-threonine</td>
			<td>Sigma, 119.12 g/mol</td>
			<td>T8625-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol anhydrous</td>
			<td>Bio-Lab Itd</td>
			<td>712022300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamic acid monosodium salts hydrate&#160;</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>G1626-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-leucine</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>855448-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol anhydrous</td>
			<td>Gadot</td>
			<td>830000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">razor blade</td>
			<td>Eddison</td>
			<td>NA</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">circular cellulose filter papers</td>
			<td>Whatman, 90 mm</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glutaraldehyde</td>
			<td>EMS (Electron Micoscopy Science), 25% in water</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde&#160;</td>
			<td>EMS, 16% in water</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium cacodylate</td>
			<td>Merck, 214.05 g/mol&#160;</td>
			<td>8.2067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride 2-hydrate</td>
			<td>Merck, 147.02 g/mol&#160;</td>
			<td>1172113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stub-aluminium mount</td>
			<td>EMS, sloted head</td>
			<td>75230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">carbon adhesive tape</td>
			<td>EMS</td>
			<td>77825-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker 37&#176;C</td>
			<td>New Brunswick Scientific Innowa42</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf table top centrifuge 5424</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:301px;">Digital Sonifier, Model 250, used with Double Step Microtip</td>
			<td>Branson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 30 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 23 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter System, 500 ml, polystyrene</td>
			<td>Cornig Incorporated</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotary Shaker - Orbitron Rotatory II</td>
			<td>Boekel</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S150 Sputter Coater&#160;</td>
			<td>Edwards</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPD 030 Critical Point Dryer</td>
			<td>BAL-TEC</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Environmental Scanning Electron Microscope</td>
			<td>XL30 ESEM FEG Philips (FEI)</td>
			<td>NA</td>
			<td></td>
		</tr>
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methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

The pellicle assay is one method to study the highly regulated and dynamic processes of B. subtilis multicellularity. Besides this, the pellicle assay is suited to test a range of either pre-starter conditions or small molecule concentrations in a single cell-culture multidish plate in one experiment. However, B. subtilis pellicle formation is sensitive to the pre-culture conditions (e.g., growth medium of the pre-culture and its growth phase), the inoculation ratio and the removal of the pre-c...

Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

methodologies-for-studying-b-subtilis-biofilms-as-model-for

Research

JoVE Journal

Immunology and Infection

15.0K Views.  Weizmann Institute of Science. This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Video: Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

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

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

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

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

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

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

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 ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...