eoe sub articles

EoE Sub Articles

1. C. tropicalis&#160;biofilm formation
<ol>
	<li>Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier (Figure 1).</li>
	<li>Add 100 &#181;L of&#160;C. tropicalis&#160;culture (from 106&#160;cells/mL stock, prepared as above) to a 96-well microtiter plate using a multichannel pipette (Figure 2A). Keep the last two columns (11 and 12) as &#39;no fungus plus serum&#39; and &#39;no fungus and no serum&#39; negative controls by not adding fungal cells. Fill columns 11 and 12 with 100 &#181;L of Roswell Park Memorial Institute (RPMI) 1640 morpholinepropanesulfonic acid (MOPS) medium alone.</li>
	<li>Cover the microtiter plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 &#176;C under stationary conditions.</li>
	<li>The next day, aspirate the medium carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual medium.</li>
	<li>Wash the plate with 200 &#181;L of 1x phosphate-buffered saline (PBS) (per well) using a multichannel pipette. Add PBS very gently along the side walls of the well to avoid disrupting biofilms. Aspirate the PBS carefully using a multichannel pipette. Repeat the PBS wash 2x (a total of three washes).</li>
	<li>To remove excess PBS, air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet.</li>
</ol>
2. Treatment of biofilm with antibodies
NOTE: Biofilms can now be processed for assessing the inhibition of biofilm maturation by antibodies. Murine serum was used as the source of polyclonal antibodies. Different groups of Sap2-immunized (Sap2-albicans, Sap2-tropicalis, and Sap2-parapsilosis) along with sham-immunized mice were bled retro-orbitally and serum was isolated. The presence of anti-Sap2 antibodies was confirmed using Sap2-specific enzyme-linked immunosorbent assay (ELISA).
<ol>
	<li>Perform heat-inactivation of the serum (source of polyclonal antibodies) at 56 &#176;C for 30 min before use to rule out the role of complement in the inhibitory activity. Heat-inactivate the serum before making serum dilution. 
	NOTE: Use serum from sham-immunized mice, preimmune mice, and Sap2-specific antibody-depleted serum as additional controls. Antibody-depleted serum was prepared as per a previous study. Among the serial dilutions (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, and 1:12,800) for serum tested in this protocol, inhibition of biofilm maturation was observed at 1:25, 1:50, and 1:100; hence, 1:50 was selected to strike a balance between inhibition and serum consumption.</li>
	<li>Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI 1640 MOPS medium (1:50). Use a common serum dilution (1:50) for all the serum samples to be tested for inhibition of biofilm maturation.</li>
	<li>Add 100 &#181;L of the selected serum dilution to each well of the 96-well microtiter plate. For each sample, add serum dilutions in duplicate, as per the layout attached (Figure 2B).
	<ol>
		<li>In column 10, do not add serum dilution; add only RPMI 1640 MOPS medium for the fungus-only positive control. 
		NOTE: Column 10, rows G1-G8 and H1-H8 initially had fungal cells in RPMI-MOPS. However, while RPMI-MOPS was added to column 10 even after 24 h, rows G1-G8 served as PBS control and rows H1-H8 as no-serum control after 24 h.</li>
		<li>In column 11, add a 1:50 dilution of serum to all wells to serve as the no fungus plus serum negative control.</li>
		<li>In column 12, do not add serum dilution to any well; keep this as the no fungus no serum negative control.</li>
	</ol>
	</li>
	<li>Cover the plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 &#176;C.</li>
</ol>
3. Biofilm metabolic activity estimation
<ol>
	<li>The next day, aspirate the serum carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual serum.</li>
	<li>Wash the plate with 200 &#181;L of 1x PBS (per well) using a multichannel pipette, adding the PBS along the side walls of the well to avoid disrupting the biofilms. Aspirate the PBS carefully using a multichannel pipette and repeat the PBS wash 2x (a total of three washes). Air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet to dry any excess PBS.</li>
	<li>Preparation of XTT(2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)/menadione:
	<ol>
		<li>Prepare XTT in sterile Ringers Lactate as a 0.5 g/L solution. Dissolve 25 mg of XTT in 50 mL of filter-sterilized Ringers Lactate. Aliquot 10 mL in separate tubes covered with aluminum foil and store at -80 &#176;C.</li>
		<li>Prepare menadione as a 10 mM stock. Dissolve 8.6 mg of menadione in 5 mL of acetone and distribute 50 &#181;L in 100 separate microtubes. Store the aliquots at -80 &#176;C.</li>
		<li>Prepare XTT/menadione solution just before use by taking 10 mL of XTT and adding 1 &#181;L of menadione to obtain a 1 &#181;M working solution.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of the XTT/menadione solution per well of the 96-well microtiter plate. Cover the plate with a lid and aluminum foil. Incubate the plate for 2 h at 37 &#176;C in the dark.</li>
	<li>Transfer 80 &#181;L of the colored supernatant from each well into a fresh 96-well plate. Read the plate at 490 nm.</li>
	<li>Calculate the mean of the absorbance values of the wells in column 10 (fungus-only positive control), which will serve as a reference value for calculating the percentage biofilm inhibition by each serum sample using equation (1). 
	% Biofilm inhibition = 100 - <img alt="Equation 1" src="/files/ftp_upload/22101/22101eq01.jpg" style="margin: auto;" /> &#215; 100&#160;&#160; &#160;(1)</li>
</ol>

an in vitro assay to evaluate the effect of antibodies on candida tropicalis biofilm

Source: Chandley, P., et al. <a href="https://app.jove.com/v/64425">A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.</a> J. Vis. Exp. (2022).
This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

<img alt="Figure 1" src="/files/ftp_upload/22101/22101_Figure1.jpg" /> 
Figure 1: Imaging&#160;Candida tropicalis&#160;biofilm.&#160;(A) Visualization of&#160;Candida tropicalis&#160;biofilm formed on the bottom of a 96-well microtiter plate after removal of RPMI medium, using an inverted microscope. The image was captured using brightfield microscopy (no stain was used). (B) Visualization of&#160;C. tropicalis<...

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

1. C. tropicalis&#160;biofilm formation

	Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier ...

Take a microplate containing the fungus Candida tropicalis. 
The cells secrete Sap2, a proteolytic enzyme that cleaves a transmembrane glycoprotein called Msb2, removing its inhibitory domain.
This induces the fungus to adhere to the bottom and grow into a microcolony of yeast and filamentous forms. The cells secrete soluble components and biopolymers, initiating biofilm formation.
Add a serum sample containing Sap2-specific antibodies to selected wells, and incubate in the dark.
The antibodies bind to Sap2 and neutralize its activity, impeding cell viability and biofilm maturation.
In untreated wells, the biofilm matures into a dense network of cells and extracellular matrix.
Aspirate the liquid, wash the biofilm, and air-dry the plate. Add a chromogenic substrate, and incubate in the dark.
Viable cells, utilizing membrane-bound oxidoreductases, reduce the substrate to a colored product.
Transfer the colored supernatant and read the absorbance.
A lower absorbance in the serum-treated wells indicates antibody-mediated biofilm inhibition.&#160;

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Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

48 Views. Source: Chandley, P., et al. A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms. J. Vis. Exp. (2022). This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofi...

Video: An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm - Concept

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

JoVE Journal

Immunology and Infection

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Medicine

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 ...

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

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