eoe sub articles

EoE Sub Articles

1. C. albicans Growth
<ol>
	<li>Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 g of yeast extract granulated, 20 g of bacteriological peptone, and 15 g of granulated agar. Makeup volume to 900 ml with Milli-Q water and autoclave.</li>
	<li>Add 50 ml of sterile 40% glucose. Mix thoroughly and pour into Petri dishes. Leave agar plates to cool and solidify. 
	NOTE: In this study, use two strains, namely wild type C. albicans SC5314 &#8211;gLuc negative strain (referred to as WT) and C. albicans SKCA23 strain, which is a wild type C. albicans SC5314 transformed with Clp10::Act1p-gLUC59 plasmid 22. In this strain (named SKCA23-ACTgLuc) gLuc was fused to the endogenous PGA59 gene under the control of the ACT1 (actin) promoter (this promoter is active in the yeast, as well as the hyphal stage of fungal growth). This strain can be requested from the laboratory of Prof. Patrick Van Dijck, KU Leuven, Leuven, Belgium. Plasmid Clp10::Act1p-gLUC59 plasmid 22 was kindly donated by Prof. C. d&#39;Enfert, Institute Pasteur, Paris, France.</li>
	<li>Maintain both strains in glycerol stock and store at -80 &#176;C.</li>
	<li>Prior to any experiment streak strains onto an YPD plate. Incubate plate at 37 &#176;C overnight.</li>
</ol>
2. Catheter Pieces Preparation
<ol>
	<li>Before the experiment, determine how many catheters are needed. It is possible to implant up to 6 catheter pieces per mouse (3 catheters on the left and 3 catheters on the right side of the animal (Figure 1A).</li>
	<li>Twenty-four hr prior to the animal surgery, open the package containing the triple-lumen catheter under the biological safety cabinet. Remove all unnecessary parts with sterile tweezers and cut the part attached to the catheter with a sterile scalpel. Place the ruler under the plastic package and cut polyurethane catheter pieces of exactly 1 cm according to the scale on the ruler (Figure 2). 
	NOTE: It is important to mention that this type of catheter piece is not phosphorescent and therefore suitable for BLI.</li>
	<li>Place a maximum of 15 cut catheter pieces (step. 2.2) into 2 ml microcentrifuge tubes. Always prepare 3 pieces extra, which are used to enumerate the amount of attached Candida cells on the device after the period of adhesion.</li>
	<li>Supplement catheters with approximately 1.8 ml of 100% fetal bovine serum (FBS).</li>
	<li>Vigorously vortex and add an additional 100-200 &#956;l of 100% FBS. Completely cover all catheter pieces with serum.</li>
	<li>Incubate at 37 &#176;C overnight.</li>
</ol>
3. Ex vivo C. albicans Adhesion on FBS-coated Polyurethane Substrates
<ol>
	<li>Transfer each serum-coated catheter piece into a fresh 1.5 ml microcentrifuge tube.</li>
	<li>Scrape off some C. albicans cells grown on YPD plates (Step 1) and suspend them in 1 ml of PBS.</li>
	<li>Dilute Candida cells (1:100) in a separate microcentrifuge tube. Take 10 &#956;l of the diluted sample and apply it to a cell counting chamber. Count at least 16 small squares.</li>
	<li>Prepare Candida cells (each strain separately) in RPMI 1640 medium to a final concentration of 5 x 104 cells/ml.</li>
	<li>Add 1 ml of cell suspension to each serum-coated catheter piece.</li>
	<li>Vigorously vortex and ensure that the catheters are submerged in the medium and not floating on top.</li>
	<li>Incubate catheters at 37 &#176;C for 90 min (period of adhesion).</li>
	<li>Remove catheters with sterile tweezers and wash them twice with 1 ml of PBS. During this step make sure that the washing fluid goes through the lumen by keeping the catheter in a vertical position while very gently flushing the catheter. Importantly, do not use a strong flow which may lead to the removal of attached cells.</li>
	<li>Transfer each washed catheter to a clean microcentrifuge tube (one piece per tube).</li>
	<li>Place on ice and keep there until surgery.</li>
</ol>
4. Anesthesia
<ol>
	<li>Prepare anesthesia by mixing 75 &#181;l of ketamine (100 mg/ml) with 100 &#181;l of medetomidine (1 mg/ml) and 825 &#181;l of sterile saline. Administer intraperitoneally (i.p.) 60-80 &#181;l of anesthetic cocktail per 10 g body weight, resulting in a dose of 45-60 mg/kg ketamine and 0.6-0.8 mg/kg medetomidine.</li>
	<li>For reversal of anesthesia, dilute 50 &#181;l atipamezole (5 mg/ml) in 4.95 ml saline, and administer i.p. 100 &#181;l per 10 g body weight as an antidote, resulting in a dose of 0.5 mg/kg.</li>
	<li>After the injection of anesthesia, place the animal into a separate cage and wait until it is fully asleep.</li>
	<li>Confirm proper anesthetization of the animal by light skin pinch and toe pinch, which do not cause any damage to the skin. Any observed movement indicates that the animal is not sufficiently anesthetized to perform surgery. If this happens, wait a couple of minutes longer until the animal does not show any signs of movement upon skin or toe pinch.</li>
</ol>
5. Animal Surgery
<ol>
	<li>Transfer the anesthetized animal from the cage on a clean tissue placed on the heating pad, pre-warmed to 37&#176;C (Figure 1A). 
	NOTE: A cheaper alternative is to use electrically heated blankets. It is also possible to use isothermic pads, which must be warmed up in the microwave prior to the animal surgery.</li>
	<li>Apply ophthalmic ointment on the eyes.</li>
	<li>Shave the lower back of the animal with an electric razor. Remove all animal hairs and transfer the animal to a clean tissue. Disinfect the skin (e.g., with 1% iodine isopropanol or 0.5% chlorhexidine in 70% alcohol) and leave the disinfected area to dry for approximately 1 min (Figure 1A).</li>
	<li>Make a small incision in the skin (one on the left and one on the right side of the animal) (approximately 0.5&#8211;1 cm) (Figure 1A).</li>
	<li>Dissect the subcutis with a scissor to create two subcutaneous tunnels. Each tunnel should be approximately 1.5 cm long and 1 cm wide.</li>
	<li>Insert three catheter pieces, previously infected with Candida, in each tunnel. Ensure that the catheters lie next to each other in a horizontal arrangement and that they do not cover each other to enable the implantation of six catheter fragments in total (Figure 1A).</li>
	<li>Close the incisions with sutures. Alternatively, use Dermabond to close the wound.</li>
	<li>Disinfect the wound very gently with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).</li>
	<li>Apply local anesthetic (xylocaine gel, 2%) directly on the wound.</li>
	<li>Administer reversal of anesthesia (protocol 5, step 2): intraperitoneally 100 &#181;l per 10 g body weight.</li>
	<li>Transfer the animal to a clean cage previously placed on a heating plate. Keep the animal separate and warm until the animal is completely awake. Meanwhile, continue with the operation and implant of the next animal. Once all operated animals are fully awake. Transfer to one cage. Monitor animals regularly.</li>
</ol>
6. Bioluminescence Imaging: Preparation of Coelenterazine (CTZ), the Substrate for G. princeps luciferase
<ol>
	<li>Prepare fresh coelenterazine (CTZ) stock solution by dissolving 5 mg/ml CTZ in acidified ethanol or according to the manufacturer&#39;s instructions.</li>
	<li>Prepare a 1.2 mM working solution by diluting the stock solution 1:10 in sterile PBS. 
	NOTE: Inject 100 &#181;l CTZ working solutions subcutaneously in the area surrounding the catheters. Use insulin syringes for subcutaneous injection of CTZ.</li>
	<li>Always keep CTZ in the dark (e.g., cover microcentrifuge tubes containing CTZ with aluminum foil). Store the stock solution at -80 &#176;C for the duration of the experiments.</li>
</ol>
7. Bioluminescence Imaging
<ol>
	<li>Initialize the BLI camera.</li>
	<li>Anesthetize the animals using an induction box. Use a gas mixture of isoflurane in oxygen, N2O/ O2, or air at 2-3%.</li>
	<li>After induction, maintain anesthesia in the induction box and the imaging chamber at 1.5-2%.</li>
	<li>Before starting the imaging session, place the imaging plate in position A, which corresponds to a field of view, FOV of 10 cm. Ensure that the right position of the anesthesia outlets and animal by placing a sleeping animal in the box.</li>
	<li>Take a few photographs until the animal is in the desired imaging position, in the FOV right under the camera.</li>
	<li>Prepare two insulin syringes each containing 100 &#181;l of the CTZ working solution.</li>
	<li>Place one animal on the bench and keep it asleep by means of a nose cone providing gas anesthesia.</li>
	<li>Bring the needles of the syringes to a place surrounding the catheters subcutaneously and inject the CTZ simultaneously on top of the catheters.</li>
	<li>Immediately after injection, place the animal on the warm plate in the camera box and start the bioluminescence image acquisition.</li>
	<li>Acquire consecutive scans with acquisition times ranging from 20 to 60 sec (depending on the signal intensity) until the maximum signal intensity is reached. During the acquisition of the next frame, measure the Bioluminescence imaging BLI signal intensity of the previously acquired frames by placing an ROI over each catheter trio and measuring the photon flux through this ROI.</li>
	<li>Repeat from step 7 for the next animal(s).</li>
	<li>After imaging, return animals to their cage. Repeat BLI during the course of an experiment for longitudinal, non-invasive follow-up of biofilm formation.</li>
	<li>Analyze the BLI data using Living Image software. Place a rectangular ROI of fixed size over each catheter trio and measure the photon flux (radiance) through each ROI. Repeat this for every animal.</li>
	<li>Report the BLI signal intensity of each catheter trio as photon flux per second. Represent the BLI data on a logarithmic scale by plotting the mean and SD of the photon flux per second for each group. Perform statistical analysis on the log10 transformed data.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast extract granulated</td>
			<td>Merck</td>
			<td>MERC1.03753.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Oxoid</td>
			<td>LP037B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar granulated</td>
			<td>Difco</td>
			<td>214530</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Prepared in the laboratory</td>
			<td>for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 with L-glutamine and without sodium carbonate</td>
			<td>Sigma</td>
			<td>R6504-1L</td>
			<td>Prepare according the protocol for Candida albicans drug susceptibility testing</td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)</td>
			<td>BBraun</td>
			<td>CV-15703</td>
			<td>CV-15703</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Fagron SAS, France</td>
			<td>611139</td>
			<td>Immunosuppressant (stock solution 10 mg/ml)</td>
		</tr>
		<tr>
			<td>Insulin syringes (0.3 ml)</td>
			<td>Terumo Myjector 29G</td>
			<td>324826</td>
			<td>For injection of coelenterazine</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td></td>
			<td>For small animals</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical tools</td>
			<td></td>
			<td></td>
			<td>Scissors, 2 pairs of tweezers, scalpel</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Leica</td>
			<td>14042321474</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin suture</td>
			<td>Johnson&#38;Johnson</td>
			<td>K890H</td>
			<td>Surgical thread, needle</td>
		</tr>
		<tr>
			<td>BLI camera (IVIS Spectrum)</td>
			<td>Perkin Elmer, Alameda</td>
			<td>IVISSPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image software</td>
			<td>Perkin Elmer, Alameda</td>
			<td>(version 4.2)</td>
			<td></td>
		</tr>
	</tbody>
</table>

candida albicans biofilm formation in an in vivo mouse model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://app.jove.com/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

<img alt="Figure 1" src="/files/ftp_upload/21797/21797_Figure1 .jpg" /> 
Figure 1: Major steps during the animal surgery. (A) The procedure of catheter implant. (1) Place the anesthetized animal on a warm pad containing paper tissue and apply the ophthalmic ointment to the eyes. (2) Shave the lower part of the back and disinfect. (3) Create two small (approx. 0.5 cm) incisions through the skin on the left and on the right side of the back. Create a subcutaneous tunne...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

Take a serum-coated catheter piece in a microcentrifuge tube.
Add Candida albicans, a common biofilm-producing fungal pathogen.
Incubate to allow interactions between surface adhesins on the fungal cells and serum proteins on the catheter, enabling fungal adherence.
Wash the catheter with a buffer to remove loosely attached cells.
Next, prepare an anesthetized immunocompromised mouse for surgery.
Make a small incision in the skin and create a subcutaneous tunnel.
Insert a catheter piece, previously infected with fungi, into the tunnel.
Suture the incision.
The fungal cells divide, forming microcolonies on the catheter. These microcolonies secrete an extracellular polymeric substance, or EPS&#160; matrix, promoting cell cohesion and surface adhesion.
EPS, a biomolecule-rich material, provides structural integrity to the biofilm and shields the microbes from the host&#39;s natural defenses.
Over time, the fungal biofilm develops a complex three-dimensional structure with channels and water-filled voids, enabling nutrient exchange, waste removal, and cell-to-cell communication.

candida-albicans-biofilm-formation-in-an-in-vivo-mouse-model

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

182 ビュー. 出典:Kucharíková, S., et al.Candida albicans: Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed to Bioluminescence Imaging. J. Vis. Exp. (2015). このビデオは、皮下組織におけるカンジダ・アルビカンスのバイオフィルム形成を研究するためのin vivoマウスモデルの開発を示しています。この実験では、カンジダ菌に感染したデバイスを動物の背中の領域に埋め込んでバイ...

ビデオ: カンジダIn vivoマウスモデルにおけるバイオフィルム形成 - 概念

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.

自動マイクロ流体デバイスを使用してカンジダ菌バイオ フィルム形成の可視化

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

免疫・感染学

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

規制カンジダバイオ フィルムの成長する赤い光で毎日光線

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

カンディダ・アルビカンスバイオフィルムの明確化とイメージング

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

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Candida albicans Biofilm Formation in an In Vivo Mouse Model