a solid phase adherence assay to assess the biofilm formation

A Solid Phase Adherence Assay to Assess the Biofilm Formation

First, label two 1.5-milliliter tubes as TSB and TSB plus BS. Then, add 1 milliliter of TSB or TSB plus BS to the respective tubes.
Next, inoculate the tubes with 20 microliters of overnight culture at a 1:50 dilution. Then, add 130 microliters of uninoculated control media to each of three wells in a sterile, clear, flat-bottomed tissue culture-treated, 96-well plate. This will be the blank control.
Add 130 microliters of inoculated culture into each of the three wells. After adding the control and the inoculated culture in the respective wells, incubate the 96-well plate for 4 to 24 hours at 37 degrees Celsius statically. Once the incubation is over, set the control well as blank on the plate reader.
Next, aspirate the culture medium from each well. Then gently wash the wells once with 200 microliters of sterile PBS. After washing, aspirate the PBS, and then, invert the plate to dry for about 20 minutes.
It&#39;s very important to ensure gentle removal of the media and gentle washing to ensure that the EPS matrix is not disturbed.
Next, add 150 microliters of 0.5% crystal violet to each of the experimental and control wells. Then, incubate the plate for five minutes at room temperature. After five minutes, wash the wells once with 400 microliters of distilled water. Add 200 microliters of distilled water to wash the wells five times. After all the five washes, invert the plate, and let it dry completely, protected from the light.
In order to obtain consistent results, it is important that the samples are thoroughly dried before proceeding.
After the plate is completely dried, use 200 microliters of 95% ethanol to destain the wells. Then leave the plate on the shaker for 30 minutes at 4 degrees Celsius in order to avoid evaporation. After 30 minutes, record the OD540 on the plate reader.

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A Solid Phase Adherence Assay to Assess Biofilm Formation

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1. Preparation of Reagents
<ol>
	<li>Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile salts in 50 mL autoclaved TSB. Filter sterilizes using a 0.22 &#181;m filter. Make fresh medium weekly.&#160;&#160;&#160;&#160;&#160; 
	NOTES: The bile salts routinely used is a 1:1 mixture of sodium cholate and sodium deoxycholate isolated from ovine and bovine gallbladders. As demonstrated previously, the presence of glucose was required for bile salt-induced biofilm formation. TSB has added glucose relative to Luria-Bertani (LB) broth; and therefore, was sufficient to induce biofilm formation in&#160;Shigella&#160;and the other enteric pathogens analyzed. Depending on the bacteria to be analyzed, different glucose concentrations or different sugar requirements might be needed.</li>
	<li>0.5% w/v crystal violet in water: Dissolve 2.5 g of crystal violet in 500 mL distilled water. Filter sterilize using a 0.22 &#181;m filter.</li>
</ol>
2. Preparation of Bacteria
<ol>
	<li>Grow overnight cultures of the bacterial strains to be tested by inoculating 3 mL of TSB with a single, well-isolated colony in a sterile culture tube. Incubate at 37 &#176;C with shaking at 225 rpm for overnight incubation (16 - 24 h).&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Strains should be restreaked from freezer stocks every 2 to 4 weeks and maintained on plates no more than 2 weeks old.</li>
</ol>
3. Solid-phase Adherence Assay
NOTE: This assay quantifies adherent bacteria using a 96-well plate method. Bacteria are grown statically in flat bottom plates. Washing is performed to remove non-adherent bacteria and adherent bacteria are stained with crystal violet. The crystal violet stain binds peptidoglycan in the bacterial cell wall and can be solubilized using ethanol. The number of adherent bacteria is determined based on crystal violet retention.
<ol>
	<li>Set up two 1.5 mL tubes. Label with TSB or TSB + bile salts (BS).</li>
	<li>Add 1 mL of TSB or TSB + BS to the respective tubes.</li>
	<li>Inoculate tubes with 20 &#181;L of overnight culture (at a 1:50 dilution).</li>
	<li>In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 &#181;L/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type (TSB and TSB + BS) to be tested.</li>
	<li>Add 130 &#181;L/well of inoculated culture into three wells and repeat until all experimental conditions are plated in triplicate.</li>
	<li>Incubate for 4 - 24 h at 37 &#176;C statically.</li>
	<li>Using a plate reader, record the OD600. Set the control wells as &#39;blank.&#39;&#160;Confirm the control medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD600&#160;values can be used to normalize the data if there are significant differences in growth rate between bacterial strains.</li>
	<li>Remove the culture medium using a vacuum line by gently tilting the plate and slowly aspirating the medium at the lower edge of the well. Be sure to collect all the culture medium without disrupting the adherent bacterial population located on the plastic surface. If extracellular polysaccharide (EPS) matrix was produced during the incubation time, the matrix will be visualized as a white precipitate. Do not disturb the EPS matrix.</li>
	<li>Gently wash the wells once with 200 &#181;L sterile phosphate-buffered saline (PBS). Remove the PBS wash using the vacuum line.</li>
	<li>Invert the plate to dry. Allow a minimum of 20 min to dry.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Since the biofilm must be thoroughly dried before staining, the protocol can be paused at this step for a few hours or even overnight. The added drying time will not alter the staining procedure while incomplete drying will affect quantification of the results and reproducibility.</li>
	<li>Add 150 &#181;L of 0.5% crystal violet to each experimental and control well.</li>
	<li>Incubate for 5 min at room temperature (RT).</li>
	<li>Wash the wells once with 400 &#181;L of distilled water. The added volume helps to remove residual crystal violet stain from the sides of the wells. Remove the wash with the vacuum line.</li>
	<li>Wash the wells five times with 200 &#181;L of distilled water. Remove the wash with the vacuum line. 
	NOTE: Thorough washing is important for quantification. When the blank wells are clear from the distilled water and do not contain any residual crystal violet stain, proceed to the next step.</li>
	<li>Invert the plate to dry, protected from light. Ensure complete drying of the plate as noted above.</li>
	<li>Destain the wells with 200 &#181;L of 95% ethanol. Incubate the plate on the shaker for 30 min. To avoid evaporation, particularly at higher temperatures, perform this step at 4 &#176;C.</li>
	<li>Using a plate reader, record the optical density at 540 (OD540).&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The wavelength for maximum absorbance of crystal violet is near 590 nm. The literature reports a range from OD540&#160;- OD595&#160;for crystal violet absorbance; thus choose a wavelength available based on the plate reader.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma</td>
			<td>C6158-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bile Salts</td>
			<td>Sigma</td>
			<td>B8756-100G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma</td>
			<td>L7533-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL culture tubes, 17 x 100 mm, plastic, sterile</td>
			<td>Fisher</td>
			<td>14-959-11B</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-bottomed 96-well plates (clear)</td>
			<td>TPP</td>
			<td>92696</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate reader</td>
			<td>Spectramax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Shaking Incubator</td>
			<td>New Brunswick Scientific Excella E25</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C Plate Incubator</td>
			<td>Thermolyne Series 5000</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a solid phase adherence assay to assess biofilm formation

Source: Nickerson, K. P. et al., <a href="https://app.jove.com/v/57322">Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification.</a>&#160;J. Vis. Exp.&#160;(2018)
The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

1. Preparation of Reagents

	Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile ...

Take a flat-bottom multi-well plate with wells containing bacteria in growth media or bacteria in growth media supplemented with bile salt.
The bile salts induce stress, causing cellular damage and releasing DNA and proteins. These biomolecules coat the well bottoms, enabling the surviving bacteria to adhere to the solid surface.
The attached bacteria multiply, forming micro-colonies and producing auto-inducers &#8212; or chemical signaling molecules, allowing bacterial communication.
This communication triggers bacteria to colonize and initiate the production of extracellular polymeric substances or EPS, generating a biofilm.
Post-incubation, remove the media to eliminate free-floating bacteria.
Introduce a crystal violet stain that interacts with the peptidoglycan of the bacterial cell wall, giving it a violet color.
Remove excess stain and dry. Overlay with ethanol to solubilize crystal violet from the bacterial cell wall, changing the solution color.
A more intense coloration in bile salt-treated wells than in untreated wells confirms biofilm formation through solid-phase adherence.

a-solid-phase-adherence-assay-to-assess-biofilm-formation

80 次观看. 用于评估生物膜形成的固相粘附测定

视频: 用于评估生物膜形成的固相粘附测定 - 实验

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

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

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.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

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

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

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.

使用PCR板深孔微孔设备生成更大的细菌生物膜生物质

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

A Solid Phase Adherence Assay to Assess Biofilm Formation

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