eoe sub articles

EoE Sub Articles

1. Preparation of Reagents
<ol>
	<li>Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile salts in 50 mL autoclaved TSB. Filter sterilize using a 0.22 &#181;m filter. Make fresh medium weekly. 
	NOTES: The bile salts routinely used is a 1:1 mixture of sodium cholate and sodium deoxycholate isolated from ovine and bovine gallbladders. As demonstrated 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 sterilizes using a 0.22 &#181;m filter.</li>
	<li>Phosphate-buffered saline (PBS) + Glucose: Dissolve 0.2 g glucose in 10 mL 1x PBS (2% w/v glucose final). Filter sterilizes using a 0.22 &#181;m filter. Make fresh on the day of use.</li>
	<li>PBS + Bile Salts: Dissolve 40 mg in 10 mL 1x PBS (0.4% w/v bile salts final). Filter sterilize using a 0.22 &#181;m filter. Make fresh on the day of use.</li>
	<li>PBS + glucose and bile salts: Dissolve 40 mg bile salts and 0.2 g glucose in 10 mL 1x PBS (0.4% w/v bile salts and 2% w/v glucose final). Filter sterilizes using a 0.22 &#181;m filter. Make fresh on the day of use.</li>
	<li>Prepare LB agar plates.</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 re-streaked from freezer stocks every 2 to 4 weeks and maintained on plates no more than 2 weeks old.</li>
</ol>
3. Dispersion Assay
NOTE: In this assay, the disassembly of biofilm through bacterial dispersion is detected. Here, mature biofilms are established and subsequently (usually the next day), the media are replaced with PBS or supplemented PBS. The supernatant component is then assessed to quantitate the number of bacteria that have dissociated from the biofilm.
<ol>
	<li>Set up two 1.5 mL tubes. Label with TSB or TSB + BS (bile salts).</li>
	<li>Add 1 mL of TSB or TSB + BS to the respective tubes.</li>
	<li>Inoculate tubes with 20 &#181;L of overnight culture (at a 1:50 dilution).</li>
	<li>In a sterile, clear, flat-bottomed, tissue culture-treated 96-well plate, add 130 &#181;L/well of uninoculated control media to three wells to serve as the blank control. Set up three control wells for each media type to be tested.</li>
	<li>Add 130 &#181;L/well of inoculated culture to three wells and repeat until all experimental conditions are plated in triplicate.</li>
	<li>Incubate the plate for 4 - 24 h at 37 &#176;C statically.</li>
	<li>Before continuing, warm the PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose to 37 &#176;C. Prepare these reagents fresh daily.</li>
	<li>Using a plate reader, record the optical density, OD600. Set the control wells as &#39;blank.&#39;&#160;Confirm the medium is clear with no evidence of turbidity. If any turbidity is detected, discard the experiment. The OD600&#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. Be sure to collect all the culture medium without disrupting the adherent population located on the plastic surface.</li>
	<li>Gently wash the wells twice with 200 &#181;L/well of sterile PBS. Remove the PBS wash using the vacuum line.</li>
	<li>Replace the wash with 130 &#181;L/well of the following into three wells each: PBS, PBS + glucose, PBS + bile salts, and PBS + bile salts + glucose. These reagents must be pre-warmed to 37 &#176;C.</li>
	<li>Incubate the plate for 30 min at 37 &#176;C.</li>
	<li>Carefully remove the plate from the 37 &#176;C incubator. Transfer the supernatants to a fresh, sterile 96-well plate.</li>
	<li>Using a 96-well plate or dilution block, prepare 10-fold (1:10) serial dilutions of the supernatant into sterile PBS.&#160;&#160;&#160;&#160; 
	NOTE: The dilution series should range from undiluted to 10-6&#160;depending on the bacterial strain to be tested. Pilot experiments can be performed to determine the optimal dilution range.</li>
	<li>Spot plate 5 &#181;L of each dilution onto LB agar using a multichannel pipette. Incubate at 37 &#176;C overnight. Count colonies on the next day and account for the dilution factor when determining the recovery colony forming units (CFU). To calculate the percent dispersion relative to the 1x PBS control (set at 100%), divide the recovery CFU from each treatment condition by the recovery CFU from the 1x PBS control sample. 
	NOTE: Routine media plates for the bacterial strain of interest can also be used. For example,&#160;Shigella&#160;can be plated on Congo red plates. Ensure the plates are dry for appropriate spot-plating techniques.</li>
</ol>

detection of biofilm disassembly through a dispersion assay

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 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

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 ...

Begin with a multi-well plate containing a bacterial biofilm &#8212; bacteria encapsulated within the protective extracellular polymeric substance or EPS.
Aspirate the medium to remove non-adherent bacteria and wash.
Treat bio-film-containing wells with either buffer alone, buffer with glucose, buffer with bile salts, or buffer with glucose and bile salts.
Buffer and glucose treatments support the release of bacterial enzymes that degrade the biofilm&#39;s EPS.
This results in the disassembly of the biofilm, releasing the bacteria.
While bile salts and a combination of bile salts and glucose treatments mimic stress conditions and retain bacteria within the protective EPS.
Transfer the supernatant from each well to a fresh multi-well plate.
Spot diluted supernatants on agar plates and incubate.
Enumerate bacterial colonies and determine the bacterial dispersion for each treatment.
A higher dispersion with buffer and glucose treatments compared to bile salts treatments indicates the disassembly of the biofilm.

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Research

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Immunology

Immunodiagnostics

Specialized Assays

68 Views. Source: Nickerson, K. P. et. al., Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification. J. Vis. Exp. (2018) 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 disass...

Video: Detection of Biofilm Disassembly Through a Dispersion Assay - Concept

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

JoVE Journal

Environment

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Assessing Biofilm Dispersal in Murine Wounds

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Here, we describe ex vivo and in vivo methods for assessing bacterial dispersal from a wound infection in mice. This protocol can be utilized to test the efficacy of topical antimicrobial and anti-biofilm therapies, or to assess the dispersal capacity of different bacterial strains or species.

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

Detection of Biofilm Disassembly Through a Dispersion Assay

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