Name: Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors (Video) | JoVE Journal
Uploaded: 2016-10-09T14:00:00.000+00:00
Duration: 10 min 17 s
Description: Learn reproducible methodologies to study small molecule biofilm inhibitors using Bacillus subtilis as a model organism. These robust techniques provide quantitative insights into inhibition mechanisms and antibiotic resistance.

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5 minutes

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 °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 °C) agar (biofilms) and add iron(III) chloride hexahydrate to a final concentration of 50 μM (pellicles) or 250 µM (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 µl 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 µg/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 °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 ΔepsH, ΔtasA). 
		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="Biofilm target evaluation; inhibition, cytotoxicity assessment flowchart; inhibitor analysis." src="/files/ftp_upload/54612/54612fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
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 °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 °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 °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 µl 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 °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° 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 µl of the unwashed pre-culture (Step 1.7) on the dried MSgg 1.5% agar plate with the help of a template — 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 °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 °C, cut the biofilm colonies into two equal parts with the help of a razor blade and the template.</li>
</ol>
<img alt="Biofilm growth experiment setup, bacterial colony on agar, centrifugation, sonication, dilution process." src="/files/ftp_upload/54612/54612fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
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 °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 µl 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 µl PBS as in step 2.3. To assess resistance to sterilizing agents, transfer the second half of the biofilm colony to 500 µl 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 µl 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 µl of PBS to a final volume of 1 ml. Perform a serial dilution (to 10-7) in PBS and spread 100 µl 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 °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 °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 °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 °C. At a temperature of 31.1 °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 °C, incubate for 10 min. At 42 °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 °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° angle in a gold-palladium sputter-coater. Repeat this step twice and rotate the samples by 120° 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><tbody><tr><td>Luria Broth, Lennox</td><td>Difco</td><td>240230</td><td></td></tr><tr><td>Bacto Agar</td><td>Difco</td><td>214010</td><td></td></tr><tr><td>potassium phosphate monobasic&amp;nbsp;</td><td>Sigma, 136.09 g/mol</td><td>P0662-500G</td><td></td></tr><tr><td>potassium phosphate dibasic&amp;nbsp;</td><td>Fisher Scientific, 174.18 g/mol</td><td>BP363-1</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino)propanesulfonic acid</td><td>Fisher Scientific, 209.27 g/mol</td><td>BP308-500</td><td></td></tr><tr><td>magnesium chloride hexahydrate&amp;nbsp;</td><td>Merck, 203.30 g/mol&amp;nbsp;</td><td>1.05833.0250</td><td></td></tr><tr><td>calcium chloride anhydrous</td><td>J.T. Baker, 110.98 g/mol</td><td>1311-01</td><td></td></tr><tr><td>manganese(II) chloride tetrahydrate</td><td>Sigma, 197.91 g/mol</td><td>31422-250G-R</td><td></td></tr><tr><td>iron(III) chloride hexahydrate&amp;nbsp;</td><td>Sigma, 270.30 g/mo)</td><td>F2877-500G</td><td></td></tr><tr><td>zinc chloride anhydrous&amp;nbsp;</td><td>Acros Organics, 136.29 g/mol</td><td>424592500</td><td></td></tr><tr><td>thiamine hydrochloride</td><td>Sigma, 337.27 g/mol</td><td>T1270-100G</td><td></td></tr><tr><td>L-tryptophan</td><td>Fisher Scientific, 204.1 g/mol</td><td>BP395-100</td><td></td></tr><tr><td>L-phenylalanine</td><td>Sigma, 165.19 g/mol</td><td>P5482-100G</td><td></td></tr><tr><td>L-threonine</td><td>Sigma, 119.12 g/mol</td><td>T8625-100G</td><td></td></tr><tr><td>glycerol anhydrous</td><td>Bio-Lab Itd</td><td>712022300</td><td></td></tr><tr><td>L-glutamic acid monosodium salts hydrate&amp;nbsp;</td><td>Sigma, 169.11 g/mol</td><td>G1626-1KG</td><td></td></tr><tr><td>D-leucine</td><td>Sigma, 169.11 g/mol</td><td>855448-10G</td><td></td></tr><tr><td>ethanol anhydrous</td><td>Gadot</td><td>830000054</td><td></td></tr><tr><td>razor blade</td><td>Eddison</td><td>NA</td><td></td></tr><tr><td>circular cellulose filter papers</td><td>Whatman, 90 mm</td><td>1001-090</td><td></td></tr><tr><td>glutaraldehyde</td><td>EMS (Electron Micoscopy Science), 25% in water</td><td>16220</td><td></td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>EMS, 16% in water</td><td>15710</td><td></td></tr><tr><td>sodium cacodylate</td><td>Merck, 214.05 g/mol&amp;nbsp;</td><td>8.2067</td><td></td></tr><tr><td>calcium chloride 2-hydrate</td><td>Merck, 147.02 g/mol&amp;nbsp;</td><td>1172113</td><td></td></tr><tr><td>stub-aluminium mount</td><td>EMS, sloted head</td><td>75230</td><td></td></tr><tr><td>carbon adhesive tape</td><td>EMS</td><td>77825-12</td><td></td></tr><tr><td>Shaker 37 &amp;deg;C</td><td>New Brunswick Scientific Innowa42</td><td>NA</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf table top centrifuge 5424</td><td>NA</td><td></td></tr><tr><td>Digital Sonifier, Model 250, used with Double Step Microtip</td><td>Branson</td><td>NA</td><td></td></tr><tr><td>Incubator 30 &amp;deg;C</td><td>Binder</td><td>NA</td><td></td></tr><tr><td>Incubator 23 &amp;deg;C</td><td>Binder</td><td>NA</td><td></td></tr><tr><td>Filter System, 500 ml, polystyrene</td><td>Cornig Incorporated</td><td>NA</td><td></td></tr><tr><td>Rotary Shaker - Orbitron Rotatory II</td><td>Boekel</td><td>NA</td><td></td></tr><tr><td>S150 Sputter Coater&amp;nbsp;</td><td>Edwards</td><td>NA</td><td></td></tr><tr><td>CPD 030 Critical Point Dryer</td><td>BAL-TEC</td><td>NA</td><td></td></tr><tr><td>Environmental Scanning Electron Microscope</td><td>XL30 ESEM FEG Philips (FEI)</td><td>NA</td><td></td></tr></tbody></table>

methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors

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...

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

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

Bacterial biofilms are important for human health, protecting their bacterial residents from environmental insults and anti-microbial agents. The goal of this procedure was to develop a model system to evaluate the effects of small molecule inhibitors on biofilm formation. These methods can help to establish a tool-set essential for the biofilm field to select small molecule inhibitors that specifically target biofilm formation.The main advantage of these methodologies is that they start from a consistent, robust experimental framework, and give quantitative and qualitative insights into the mechanism of action of biofilm-specific inhibitors. Though these methods can provide insight into Bacillus subtilis biofilms, they can also be applied to other biofilm-forming bacteria, such as Pseudomonas aeruginosa or the plant pathogen Xanthomonas citri. Scanning electron microscopy can be essential to analyze the effect of small molecules on biofilm formation, because it allows observation of the extra-cellular matrix, and gives single-cell resolution.To begin, first select an appropriate single colony, and transfer it into three-milliliters of LB broth. Place this starter culture into a shaking incubator for four hours at 37 degrees Celsius. After incubation, take 1.5 milliliters of the starter culture, and centrifuge for four minutes.Carefully remove the supernatant, and then re-suspend the pellet in 1.5 milliliters of MSgg medium. To grow pellicles, prepare a 12-well cell culture plate by adding three milliliters of MSgg to each well. To some of these wells, add MSgg with a small molecule inhibitor in a concentration range, distributing the location of different concentrations around the dish, to avoid edge effects.Measure the optical density at 600 nanometers of the re-suspended starter culture. The culture should be between 0.6 and one. This is critical for the robustness of the system.Innoculate each well of the culture plate with 3 microliters of the re-suspended starter culture. Then, incubate the plate at 23 degrees Celsius for three days under static conditions. Afterwards, remove the plate from the incubator, and observe the pellicles.To grow biofilms, use a template and symmetrically spot four separate drops of 1.5 microliters of unwashed starter culture onto an 8.5 centimeter dried 1.5%Agar MSgg plate. Let the drops absorb into the medium before moving the plate. Incubate the plates at 30 degrees Celsius for three days.Using a binocular with homogeneous exposure of lighting, check that biofilm colonies have developed and formed a three-dimensional wrinkled structure. First, take a clean razor blade and cut the biofilm colonies into two equal parts with the help of the template. Carefully lift one half of the colony with a clean spatula, and transfer it to a 1.5-milliliter micro-centrifuge tube, containing 500 microliters of PBS.Take the second half of the colony, and transfer it to a micro-centrifuge tube containing 500 microliters of 50%ethanol. These will be used to assess resistance to sterilizing agents. Afterwards, similarly take the first half of the D-lysine treated colony, and place it in PBS.Then, take the second half of this colony, and transfer it to ethanol. Incubate all the tubes containing the biofilm halves for 10 minutes at room temperature. And then, centrifuge them for five minutes at 18, 000 times g.Using a pipette, carefully remove the supernatant. Add 300 microliters of PBS, and then sonicate the cells at a low setting, with a microtip sonicator. Add an additional 700 microliters of PBS for a final volume of one milliliter.Next, perform a serial dilution of 10 to the negative seven in PBS. Take 100 microliters of one of the dilutions, and innoculate a 1.5%Agar LB plate. Repeat this step for two other dilutions per sample.Spread the innoculum using glass beads, and then incubate the plates overnight at 30 degrees Celsius. Examine the plates and count the colony-forming units, or CFU. After calculating the CFU per milliliter value, calculate the percentage of survivors in the PBS versus ethanol treatments.To begin sample fixation, first prepare enough fresh fixative solution for the desired number of biofilm colonies. Carefully add five milliliters of fixative to each petri dish, and avoid pipetting directly onto colonies. Colonies will begin to detach from the Agar and float.Carefully seal the plates with parafilm, and incubate on a rotary shaker for two hours at room temperature. Transfer the plates to four degrees Celsius storage overnight. Gently remove the fixative liquid with a Pasteur pipette, connected to a vacuum.Add 10 milliliters of 100 nanomolar sodium cacodylate, five millimolar calcium chloride buffer to wash the biofilm, and incubate for five minutes. Carefully detach the center of the biofilm colony from the Agar plate with a Pasteur pipette. To air-dry colonies, cut cellulose filter paper into quarters, and then submerge one section in 100%ethanol.Carefully transfer one floating biofilm colony onto the paper. Place the paper in a Petri dish lined with dry filter paper. Then, cover the dish, and leave it in a chemical hood to dry overnight.In order to preserve the morphology of the biofilm colony, it is important to underlay the floating biofilm completely with the cellulose paper. Once on the paper, the biofilm cannot be readjusted. Coat an electron microscopy stub with carbon tape, and then use tweezers to carefully transfer the biofilm colonies onto the stub.After connecting each colony to the stub, by adding a thin bridge of carbon tape, store the stubs in a desiccator for 24 hours or until needed. This step requires a steady and precise hand, because at this stage the biofilms are very fragile and easily fractured. The challenge is to mount a substantial part of the biofilm without cracks.On the day of the examination, place the colonies into a gold-palladium sputter coater. Coat the samples for two minutes at a 60 degree angle. Repeat this step twice, rotating samples 120 degrees in between.Finally, coat the samples once for three minutes from the top. Samples are now ready for imaging on the SEM. These images show pellical formation of B.subtilis grown in biofilm-inducing MSgg medium.With the addition of the small molecule inhibitor D-lysine, pellicle development is reduced. And as the concentration of the inhibitor increases, pellicle formation shows a correlating decrease. This graph shows the effect of exposure to ethanol on the survival of cells within biofilm colonies, treated with a D-lysine inhibitor, or left untreated.Colonies treated with D-lysine, and exposed to 50%ethanol for 10 minutes, showed a dramatic reduction in survival compared to the untreated fraction. Top-down binocular images show that colonies grown in the presence of D-lysine were smaller overall, and formed less pronounced wrinkles than those left untreated. Scanning electron microscope images of critical point dried biofilm colonies reveal yet further the altered biofilm development.The extra-cellular polymeric substances matrix, or EPS, appeared more like a spider web in untreated samples, with treated samples showing less organization. Finally, examination at the resolution of individual bacterial cells shows that treated cells are elongated in appearance, less covered with EPS, and not as tightly connected to their neighbors as the untreated cells. While attempting this procedure, it's important to remember that setting a consistent framework to study biofilm inhibitors is essential for reproducible results.Once mastered, a small molecule screening for biofilm inhibition can be done in less than a week if it's performed properly. Following this procedure, other methods, such as investigation of gene expression in single biofilm cells, can be performed in order to obtain additional insights into the target of the small molecule inhibitor. After watching this video, you should have a good understanding of how to analyze the overall effect of specific biofilm inhibitors on biofilm development and antibiotic resistance.

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Immunology and Infection

Source: Tay, S. B., et al. Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases. J. Vis. Exp. (2016)The video demonstrates a method to quantify biofilm biomass in a pathogenic bacterium, comparing untreated controls with samples treated with a quorum-quenching enzyme. The enzyme disrupts bacterial signaling during early incubation, reducing cell adhesion. After washing and further incubation, control wells form dense biofilms, while treated wells show...

Quantifying Enzyme-Induced Biofilm Suppression in Pathogenic Bacteria

Source: Tay, S. B., et.al. Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases. J. Vis. Exp. (2016)This video demonstrates the process of inhibiting biofilm formation by introducing an enzyme that disrupts bacterial cell signaling. The enzyme prevents the bacteria from producing extracellular polymeric substances, thereby reducing biofilm development on a glass-bottomed microdish. Finally, the biofilm structure is visualized using fluorescent lectins and...

Microscopic Assessment of Enzyme-Mediated Inhibition of Biofilm Formation

Source: Bucher, T., et al. Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp. (2016)This video demonstrates a pellicle formation assay using Bacillus subtilis to evaluate small-molecule inhibitors that disrupt matrix synthesis and biofilm development, providing a model to screen compounds that selectively inhibit surface-associated growth without affecting free-floating cells.

Evaluating Inhibitor Effects on Pellicle Formation in Bacillus subtilis

Source: Bucher, T., et al. Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp. (2016).This video demonstrates how biofilm matrix integrity influences bacterial resistance to ethanol by comparing colony-forming units in untreated and inhibitor-disrupted biofilms. Reduced CFUs in inhibitor-treated samples demonstrate that the extracellular matrix is essential for protecting biofilm-embedded cells from antimicrobial stress.

Ethanol Resistance Assay to Evaluate Biofilm Matrix Integrity

Source: Bucher, T., et. al., Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp.(2016)This video demonstrates the preparation of mature bacterial biofilm colonies for high-resolution imaging. It covers fixation, dehydration, mounting, and metal coating steps to preserve and visualize biofilm morphology.

Preparation of Bacterial Biofilm Colonies for Morphological Analysis

The video showcases a biofilm dispersion assay using a multi-well plate. Buffer and glucose treatment disassemble the biofilm, releasing bacteria, while bile salts induce stress, retaining bacteria in the protective extracellular polymeric substance. Higher dispersion with buffer and glucose confirms successful biofilm disassembly, estimated by colony forming units measurement.

Detection of Biofilm Disassembly Through a Dispersion Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

Learn reproducible methodologies to study small molecule biofilm inhibitors using Bacillus subtilis as a model organism. These robust techniques provide quantitative insights into inhibition mechanisms and antibiotic resistance.

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

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