The authors acknowledge resources and support from Midwestern University Cellular and Molecular Core Research facility.

All animal protocols were approved by Midwestern University Institutional Animal Care and Use Committee (IACUC) as described before57. The Institutional Animal Care and Use Committee at Midwestern University approved this study under MWU IACUC Protocol #2894. The MWU animal care policies follow the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals and the policies laid out in the Animal Welfare Act (AWA).
1. Mice study standard protocol
<ol>
	<li>Use male and female C57BL/6J mice at least six weeks old. Supplement them with sterile water with or without cefoperazone (0.5 mg/mL).
	<ol>
		<li>Co-house mice in groups of 5, with each cage containing either all male or all female mice. Provide the mice standard mouse chow and water (via a 400 mL bottle) at all times.</li>
		<li>Check cages daily to ensure food and water levels are enough, and to examine mice for signs of distress.</li>
	</ol>
	</li>
	<li>Replace the water with cefoperazone every 48 h to ensure fresh antibiotic is being provided regardless of remaining water in the cage feeding bottles.</li>
	<li>After 5&#8210;7 days of cefoperazone treatment, euthanize mice via CO2 asphyxiation observing established IACUC protocol. Confirm death via cervical dislocation.</li>
	<li>Dissect mice using autoclave-sterilized sharp ended scissors and autoclave-sterilized forceps.
	<ol>
		<li>After euthanasia, secure the animal&#160;to a dissection surface by pinning all limbs such that the abdomen is exposed.</li>
		<li>Spray the abdominal region with 70% ethanol to prevent fur from sticking to forceps, scissors, or gut sections during dissection.</li>
		<li>Use forceps to pinch and lift a section of skin at the base of the abdomen and create a small incision through the skin and underlying fascia using scissors. Take great care when making this incision to avoid puncturing the cecum or intestinal wall.</li>
		<li>Extend this cut to the rib cage, partially exposing the peritoneal cavity. Make a cut starting at the point of the initial incision on either side extending upward and laterally.</li>
		<li>Pull these flaps laterally and pin to the dissecting surface to fully expose the peritoneal cavity.</li>
	</ol>
	</li>
	<li>Extract the GI tract using forceps, while using scissors to make cuts superior to the stomach and at the distal region of the large intestine to ensure collection of the greatest amount of gut content from each section.</li>
	<li>When removing the GI tract, take care to avoid rupturing the individual components. Separate the stomach, small intestine, cecum, and large intestine individually using scissors at their proximal and distal ends.</li>
	<li>For collection of each gut contents from each section, make a single incision at the distal end of each section using scissors, followed by manually expelling the gut content into a 1.5 mL microcentrifuge tube using forceps.</li>
	<li>Store gut contents at -80 &#176;C for ex vivo assays.</li>
</ol>
2. Preparation of yeast extract-peptone-dextrose (YPD) agar plates
<ol>
	<li>To a 1 L glass bottle add 25 g of yeast extract peptone-dextrose broth powder, 10 g of agar, and ultrapure water to a final volume of 500 mL.</li>
	<li>Autoclave at 121 &#176;C for 30 min on a liquid cycle to sterilize the media.</li>
	<li>Under a laminar flow hood, pour approximately 20 mL of agar media into a sterile Petri plate. 500 mL of agar media should yield approximately 25 plates.</li>
	<li>Store plates at 4 &#176;C until ready for use.</li>
</ol>
3. Ex vivo prep for hyphal morphogenesis assay
<ol>
	<li>Streak a fresh culture of C. albicans SC5314 onto a YPD agar plate and incubate overnight at 30 &#176;C.</li>
	<li>Pick two to three medium-sized individual colonies from overnight grown C. albicans SC5314 culture and re-suspend in 1 mL of phosphate buffered saline (PBS).</li>
	<li>Retrieve frozen gut contents from the -80 &#176;C freezer and thaw at 25 &#176;C.</li>
	<li>Weigh about 150 mg of gut contents into a new 1.5 mL tube.</li>
	<li>Re-suspend the gut contents with 150 &#181;L of PBS (gut content and PBS at a 1:1 weight to volume ratio).</li>
	<li>Vortex at high speed for 30 s to homogenize the gut contents and allow to sit at room temperature for about a minute.</li>
	<li>Centrifuge the homogenates at 1000 x g for 3 min.</li>
	<li>Transfer the supernatant to a new 1.5 mL tube.</li>
	<li>Repeat steps 3.7 and 3.8 to remove all debris in the supernatant.</li>
	<li>Add 10 &#181;L of the C. albicans SC5314 inoculum prepared above to this supernatant</li>
	<li>Mix well and incubate at 37 &#176;C for 4 to 5 h.</li>
</ol>
4. Exogenous addition of metabolites to the gut homogenate extracts for the hyphal morphogenesis assay
<ol>
	<li>Retrieve frozen gut contents from the -80 &#176;C freezer and re-suspended in PBS at 1:1 ratio (weight: volume).</li>
	<li>Add desired concentration of gut metabolites to the gut content and PBS mixture.</li>
	<li>Vortex at high speed for 30 s to homogenize the gut contents containing metabolites and allow to sit at room temperature for about 10 min.</li>
	<li>Centrifuge the homogenates at 1000 x g for 3 min.</li>
	<li>Transfer the supernatant to a new 1.5 mL tube. Repeat steps 4.4 and 4.5 to remove all debris in the supernatant.</li>
	<li>Add 10 &#181;L of the C. albicans SC5314 inoculum prepared above to this supernatant. Mix well and incubate at 37 &#176;C for 4 to 5 h.</li>
</ol>
5. C. albicans morphogenesis assay (immunostaining and imaging)
<ol>
	<li>Centrifuge the samples at 1000 x g for 2 min and discard the supernatant via pipetting.</li>
	<li>Fix the samples in 100 &#181;L of 2% paraformaldehyde (PFA) for 15 min.</li>
	<li>Centrifuge at 1000 x g for 2 min and discard supernatant via pipetting.</li>
	<li>Wash the samples twice with 1 mL of PBS. To wash samples, re-suspend the pellet in PBS by pipetting gently. Do not vortex the sample as this can damage hyphal structures. After re-suspension, centrifuge at 1000 x g for 2 min and discard the supernatant via pipetting.</li>
	<li>Incubate the samples at room temperature in 100 &#181;L of PBS containing polyclonal C. albicans antibody (1:100 dilution) for 30 min.</li>
	<li>Wash the samples three times with 1 mL of PBS. 
	NOTE: When using a fluorescent antibody, it is recommended that all dilution and wash steps be performed in dim light to avoid photo bleaching and improve sample longevity.</li>
	<li>Incubate the samples at room temperature for 15 min in 100 &#181;L of PBS containing anti-Rabbit IgG Alexafluor 488 antibody at 1:500 dilution. Perform incubation in a dark drawer or room to avoid photo bleaching.</li>
	<li>Wash the samples three times with 1 mL of PBS.</li>
	<li>Re-suspend the samples in 100 &#181;L of PBS and transfer to a 96-well plate for imaging. 
	NOTE: When not being imaged, it is recommended that the 96-well plate be wrapped in aluminum foil to avoid photo bleaching.</li>
	<li>Image fungal cells using 20x and 40x objective lenses using a fluorescence imaging microscope. Use a green fluorescent protein (GFP) filter (excitation wavelength 470/40 and emission wavelength 525/50) to detect fluorescence.</li>
</ol>

The authors have no competing financial interests or other conflicts of interest.

The method described here presents a novel way to investigate the effect of antibiotic, dietary, xenobiotic and therapeutic impacts on C. albicans hyphal morphogenesis in the GI tract. Since the majority of systemic infections originate from the GI tract21,22,23,24,25,26,27,</s...

Candida albicans is an opportunistic, polymorphic fungal pathogen that is normally commensal, but can undergo a morphological change into a virulent form capable of causing life-threatening infections in immunocompromised individuals1,2,3,4,5,6,7,8,9,10,11,12,13. C. albicans is a leading cause of systemic nosocomial infections, with a 40&#8210;60% mortality rate even with antifungal treatment2,14,15. Though C. albicans resides in different host niches including the female reproductive system16,17, the oral cavity of healthy individuals18 and the gastrointestinal (GI) tract19,20, the majority of the systemic infections originate from the GI tract and furthermore, the source of systemic infection is often confirmed to be the GI tract21,22,23,24,25,26,27,28,29,30,31,32,33,34. C. albicans pathogenicity in the GI tract is influenced by a wide range of factors; however, a major characteristic necessary for virulence is the transition from a yeast cell morphology into a virulent hyphal cell morphology35,36,37,38,39,40,41,42,43,44. C. albicans attachment and dissemination from the GI tract during infection is highly associated with its capacity to transition from a commensal yeast into virulent hyphae, allowing the fungi to cause invasive disease44,45,46,47,48,49,50,51,52,53.
A variety of factors in the gut, including n-acetylglucosamine, regulate hyphal formation by C. albicans. Therefore, it is crucial to narrow the gap in knowledge regarding the hyphal morphogenesis of this fungal pathogen in the GI tract54,55,56. Recent evidence indicates that various gut metabolites differentially control the hyphal morphogenesis of C. albicans in vitro57,58,59,60. However, technical constraints present issues when attempting to study C. albicans hyphae formation in in vivo gut samples, especially staining yeast and hyphae cells and quantitative analysis of hyphal development. To understand C. albicans hyphal morphogenesis in the GI tract, an ex vivo method was developed using soluble extracts of homogenized gut content from mice to study the effect of metabolites on fungal hyphal morphogenesis. Utilizing gut samples from mice that are resistant and susceptible to C. albicans GI infection, this method will help to identify and study the effect of metabolites, antibiotics and xenobiotics on fungal hyphal morphogenesis in the GI tract.

<table>
	<tbody>
		<tr>
			<td>1 - 10 &#181;L Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-454</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>100 - 1000 &#181;L Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-400</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>20 - 200 &#181;L Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-451</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>2-methylbutyric acid</td>
			<td>Sigma</td>
			<td>193070-25G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>488 goat anti-rabbit IgG</td>
			<td>Invitrogen (Fisher)</td>
			<td>A11008</td>
			<td>IF Staining secondary ab</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
			<td>YPD agar component</td>
		</tr>
		<tr>
			<td>Automated Imaging Microscope</td>
			<td>Keyence</td>
			<td>BZX700</td>
			<td></td>
		</tr>
		<tr>
			<td>Candida Albicans Antibody</td>
			<td>Invitrogen (Fisher)</td>
			<td>PA1-27158</td>
			<td>IF Staining primary ab</td>
		</tr>
		<tr>
			<td>cefoperazone</td>
			<td>Cayman</td>
			<td>16113</td>
			<td>antibiotic</td>
		</tr>
		<tr>
			<td>deoxycholic acid</td>
			<td>Sigma</td>
			<td>30960</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher</td>
			<td>D16-500</td>
			<td>hyphal-promoting compound</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fisher</td>
			<td>08-885</td>
			<td></td>
		</tr>
		<tr>
			<td>lactic acid</td>
			<td>Alfa Aesar</td>
			<td>AAAL13242-06</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>lithocholic acid</td>
			<td>Sigma</td>
			<td>L6250-10G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>palmitic acid</td>
			<td>Sigma</td>
			<td>P5585-10G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Alfa Aesar</td>
			<td>A11313</td>
			<td>IF Staining fixative</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS), 10x</td>
			<td>Alfa Aesar</td>
			<td>J62692</td>
			<td>PBS component</td>
		</tr>
		<tr>
			<td>p-tolylacetic acid</td>
			<td>SCBT</td>
			<td>sc-257959</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>sebacic acid</td>
			<td>Sigma</td>
			<td>283258-250G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>sharp ended scissors</td>
			<td>Fisher</td>
			<td>28301</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile Milli-Q water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>YPD Broth</td>
			<td>BD Biosciences</td>
			<td>242810</td>
			<td>YPD agar component</td>
		</tr>
	</tbody>
</table>

an ex vivo assay to study candida albicans hyphal morphogenesis in the gastrointestinal tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

These results along with previous findings from the Thangamani laboratory60 indicate that when C. albicans is grown ex vivo in gut homogenate extracts taken from the stomach, small intestines and large intestines of untreated control and antibiotic-treated mice, C. albicans generally develops with a yeast morphology (Figure 1B). However, when grown in the cecal extract from antibiotic-treated mice, C. albicans readily undergoes morphogenesis...

Watch this Scientific Journal Video about An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract at JoVE.com

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

an-ex-vivo-assay-to-study-candida-albicans-hyphal-morphogenesis

Research

JoVE Journal

Immunology and Infection

5.2K Views.  Midwestern University. The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Video: An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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. ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

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.

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 ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

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.

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

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...