We thank Dr. Paula da Silveira, Dr. Cec&#237;lia Atem Gon&#231;alves de Ara&#250;jo Costa, Shawn M. Maule, Shane M. Maule, Dr. Malvin N. Janal and Dr. Iriana Zanin for the development of this study. We also acknowledge Dr. Alexander D. Johnson (UCSF) for donating the strain analyzed in this study.

1. Preparation of culture media
<ol>
	<li>Prepare sabouraud dextrose agar (SDA). Suspend 65 g of SDA supplemented with chloramphenicol (50 mg/L) in 1,000 mL of distilled water. Boil to dissolve the medium completely. Sterilize by autoclaving at 15 PSI (121&#176;C) for 30 min. Cool to 45-50 &#176;C. Mix well and pour 20 mL of SDA into sterile Petri plates (size: 100 mm x 15 mm).</li>
	<li>Prepare yeast nitrogen base (YNB) medium supplemented with 100 mM glucose by mixing 6.7 g of YNB and 18 g of dextrose to 1,000 mL of ultrapure water. Mix using a stirrer and sterilize the medium using a 0.22 &#181;m vacuum filter system.</li>
	<li>Prepare RPMI by mixing 10.4 g of RPMI 1640 and 34.32 g of 3-(N-morpholino)propanesulfonic acid (MOPS) to 1,000 mL of ultrapure water. Mix in a stirrer without heat and adjust the pH to 7 by adding 1 N NaOH (Add 2 g of NaOH to 50 mL of ultrapure water). Sterilize the medium using a 0.22 &#181;m vacuum filter system.</li>
</ol>
2. Pre-inoculum and inoculum
<ol>
	<li>Remove the microorganism C. albicans SN425 from the &#8722;80 &#176;C freezer and let it thaw at ambient temperature. Seed 10 &#181;L of the stock culture on Petri dishes containing SDA supplemented with chloramphenicol (50 mg/L). Incubate the Petri dishes aerobically at 37 &#176;C for 48 h.</li>
	<li>After 48 h of incubation, add 10 colonies of C. albicans to 10 mL of yeast nitrogen base (YNB) medium supplemented with 100 mM glucose, and incubate aerobically at 37 &#176;C for 16 h for the pre-inoculum.</li>
	<li>After incubating for 16 h, prepare the inoculums by diluting the starter cultures into fresh YNB medium supplemented with 100 mM glucose in a 1:10 proportion (1 mL of the starter culture diluted in 9 mL of YNB). Measure the initial optical density (OD) at 540 nm.</li>
	<li>Incubate the inoculums aerobically at 37 &#176;C for 8 h until the strains reach the mid-log growth phase and measure the final OD at 540 nm. Subtract the final OD from the initial OD to check if the OD of the inoculums are on the mid-log growth phase (8 h, based on the growth curves in Figure 1).</li>
	<li>To reach an inoculum with 107&#160;cells mL-1, centrifuge the inoculum for 5 min at 5,500 x g, discard the supernatant and concentrate the inoculum to half of the volume of the tubes.</li>
</ol>
3. Biofilm formation and phototherapy
<ol>
	<li>Add 1 mL of the inoculum to the wells of a 24-well polystyrene plate. Incubate aerobically at 37 &#176;C for 90 min to allow cell adhesion to the bottom of the wells.</li>
	<li>Use a non-coherent red light (see Table of Materials) as the light source with wavelength of 635 &#177; 10 nm and a fixed output power of 1,460 mW cm&#8722;2.&#160;</li>
	<li>Use a power meter to measure the power density of the light. The radiant exposure applied is 87.6 J cm-2, equivalent to about 1 min of exposure. Use the formula Energy density (J/cm2)= power density (W/cm2) x irradiation time (s). Apply a distance of 5 mm between the light source and the biofilm to avoid warming the sample.</li>
	<li>Treat the positive control biofilms with 0.12% chlorhexidine for 1 min and negative control biofilms with 0.89% NaCl for 1 min.&#160;</li>
	<li>After treatments, wash all the biofilms twice with 0.89% NaCl solution.</li>
	<li>Add 1 mL of RPMI 1640 buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7 to the biofilms and incubate the plates at 37 &#176;C overnight.</li>
	<li>In the morning, apply the same treatments as steps 3.2 to 3.6, wash the biofilms twice with 0.89% NaCl, add new RPMI buffered with MOPS (1 mL, pH 7) to the wells and incubate aerobically at 37 &#176;C.</li>
	<li>At the same day, after 6 h, expose the biofilms to red light. Treat the positive control biofilms with 0.12% chlorhexidine for 1 min and negative control biofilms with 0.89% NaCl for 1 min. After&#160; treatments, wash the biofilms twice with 0.89% NaCl solution.</li>
	<li>Repeat the steps 3.2-3.8 until achieving 48 h of biofilm development.&#160; 
	NOTE: Basically, one treatment will happen in the morning and the other one will happen in the afternoon on the same day (6 h apart).</li>
</ol>
4. Processing
<ol>
	<li>Add 1 mL of sterile 0.89% NaCl to the well to remove the biofilm by scratching it from the well using a pipette tip. Add the removed biofilm to a sterile tube.</li>
	<li>Add another 1 mL of sterile 0.89% NaCl to the well. Scratch it again and add the suspension to the same tube for a total volume of 2 mL.</li>
	<li>Colony-forming units (CFU)
	<ol>
		<li>Vigorously vortex the biofilm suspension for 1 min. From the biofilm suspension, use an aliquot of 0.1 mL for the serial dilution.</li>
		<li>Dilute the biofilm suspension from 10-1 to 10-4 in 0.89% NaCl solution.</li>
		<li>Seed 50 &#956;L of each dilution to SDA plates and incubate the plates at 37 &#176;C for 48 h.</li>
		<li>Count the colonies and apply the formula CFU/mL = number of colonies x 10n / q (n= dilution; q= seeded volume).</li>
	</ol>
	</li>
	<li>Dry weight
	<ol>
		<li>Weigh and label microcentrifuge tubes.</li>
		<li>From the biofilm suspension, use 0.1 mL for dry weight (biomass) determination.</li>
		<li>Add 1 mL of absolute ethanol to an aliquot of 0.1 mL of the biofilms and store it at -20 &#176;C for 18 h. After 18 h, centrifuge at 10,000 x g for 10 min.</li>
		<li>Discard the supernatant, place the tubes on a desiccator to dry the samples for one week&#160;and weigh the microcentrifuge tubes again.</li>
	</ol>
	</li>
	<li>Water-soluble polysaccharides (WSPs) and alkali-soluble polysaccharides (ASPs)
	<ol>
		<li>From the biofilm suspension, vigorously vortex the remainder of the volume (1.8 mL) for 1 min and centrifuge at 5,500 &#215; g for 10 min at 4 &#176;C. Store the supernatant in a new tube and wash the precipitate twice with the cells, which contain the insoluble constituents of the extracellular matrix with sterile ultrapure water (5,500 &#215; g, 10 min at 4 &#176;C).</li>
		<li>Re-suspend the insoluble content at 1.8 mL of sterile ultrapure water.</li>
		<li>Water-soluble polysaccharides (WSPs) 
		NOTE: Perform this experiment at a fume hood.
		<ol>
			<li>From the stored supernatant, reserve 1 mL for the quantification of water-soluble polysaccharides (WSPs).</li>
			<li>Homogenize the stored supernatant for 1 min. Then, transfer to sterile tubes and add 2.5 volumes of 95% ethanol. Store the tubes for 18 h at -20 &#176;C for WSPs precipitation.</li>
			<li>After 18 h, centrifuge the tubes at 9,500 x g for 20 min at 4 &#176;C. After centrifugation, discard the supernatants.</li>
			<li>Wash the samples three times with 75% ethanol and air-dry the pellets. Re-suspend the pellet with 1 mL of sterile ultrapure water, and quantify the WSP using the phenol-sulfuric acid method.</li>
			<li>Use 0.1% glucose (0.01 g of glucose to 10 mL of ultrapure water) for the standard curve (0, 2.5, 5, 10, 15, 20, and 25 &#181;L of glucose per tube).</li>
			<li>Add 200 &#181;L of 5% phenol (2.7 mL of 90% phenol to 47.3 mL of ultrapure water) to a glass tube comprising 200 &#181;L of the sample or standard curve point (in triplicate per sample) and mix cautiously.</li>
			<li>Add 1 mL of sulfuric acid to the tube and mix. Wait 20 min for the reaction and measure the samples using a spectrophotometer (490 nm).</li>
		</ol>
		</li>
		<li>Alkali-soluble polysaccharides (ASPs) 
		NOTE: Perform this experiment in a fume hood.
		<ol>
			<li>From the insoluble re-suspended amount, separate 1 mL for the determination of ASPs. Centrifuge the aliquots of each biofilm suspension (13,000 x g, for 10 min at 4 &#176;C).</li>
			<li>Cautiously remove the supernatant of each tube. Then place the samples on a vacuum concentrator to dry the pellets.</li>
			<li>Weigh the pellets and add 300 &#181;L of 1 N NaOH (2 g of NaOH to 50 mL of ultrapure water) per 1 mg of the dry weight. Incubate them for 2 h at 37 &#176;C and then centrifuge at 13,000 &#215; g for 10 min.</li>
			<li>Cautiously collect the supernatants with a pipette and transfer to microcentrifuge tubes, maintaining the pellet.</li>
			<li>Once again, add an equal volume of 1 N NaOH to the tubes comprising the pellets, and repeat the same steps as above for the extraction of ASP.</li>
			<li>After incubation for 2 h, centrifuge the samples (13,000 x g for 10 min), carefully collect the supernatants and add to the previously collected supernatants.</li>
			<li>For the third extraction, repeat the same steps as above, but this time, do not incubate the samples for 2 h before centrifugation.</li>
			<li>Afterwards, add three volumes of 95% ethanol to each sample. Then, stock the samples at &#8722;20 &#176;C for 18 h for precipitation of ASP.</li>
			<li>Centrifuge the tubes (9,500 &#215; g for 20 min at 4 &#176;C) and discard the supernatants.</li>
			<li>Wash each pellet three times with 75% ethanol and air-dry (same procedures did for WSP). Re-suspend the pellets in equal total volume of the first extraction with 1 N NaOH.</li>
			<li>Read the samples for quantification of ASP using the phenol-sulfuric (same as for WSP analysis).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The most critical steps for successful culturing of C. albicans biofilm are: 1) to do the pre-inoculum and the inoculum in YNB medium complemented with 100 mM glucose; 2) to wait 90 min for the adhesion phase and carefully wash twice the wells with 0.89% NaCl to remove non-adhered cells; and 3) to add RPMI medium to the adhered cells to start biofilm formation, since RPMI will stimulate hyphae growth.&#160;Aneuploidies can occur when culturing C. albicans. Consequently, it is important not to use coloni...

The increased incidence of diabetes, immunosuppressive therapy applications, HIV infection, AIDS epidemic, invasive clinical procedures and broad-spectrum antibiotic consumption in the past years have increased the incidence of Candida albicans related diseases1,2. C. albicans infections are commonly related to biofilm development and may cause clinical manifestations, such as candidiasis, or systemic manifestations, such as candidemia1,2. One of the most noteworthy virulence factors of biofilm growth is the extracellular polysaccharide matrix establishment. Biofilm formation cooperates to increase the resistance to existing antifungal drugs, environmental stress, and host immune mechanisms3.
The biofilm growth of C. albicans begins with the early adherence of planktonic cells to a substrate, followed by the propagation of yeast cells through the substrate surface, and hyphal growth. The last phase of biofilm growth is the maturation phase, wherein yeast-like development is suppressed, the hyphal development expands, and the extracellular matrix encloses the biofilm4. C. albicans exopolysaccharides (EPS) in the matrix interact to form the mannan-glucan complex5,6. The interaction of exopolysaccharides is critical for the defense of the biofilms against drugs7. Hence, the reduction of EPS from the C. albicans extracellular matrix could support the development of new antibiofilm protocols for oral candidiasis control.
Light regulates the growth, development, and behavior of several organisms8 and it has been applied as an antimicrobial in photodynamic antimicrobial chemotherapy (PACT). PACT applies a visible light of a specific wavelength and a light-absorbing photosensitizer9. However, the photosensitizers have difficulties in penetrating the biofilm, causing lower efficacy10. The failure of therapeutic agents to fully infiltrate biofilms is a reason that biofilms occasionally resist traditional antimicrobial therapy3,5. To deactivate the enclosed microbial cells, antimicrobials need to permeate through the extracellular matrix; nevertheless, the EPS characterizes a diffusional obstacle for such molecules by prompting their level of carriage into the biofilm or by influencing the response of the antimicrobial with the matrix itself11.
Considering the disadvantages of PACT, the use of light by itself emerges as a valuable improvement. Preliminary data revealed that the treatment with blue light twice a day significantly inhibited the production of EPS-insoluble in Streptococcus mutans biofilm. By the decrease of EPS-insoluble, blue light diminished biofilm growth. Nevertheless, the outcomes of phototherapy using red light in C. albicans biofilms are scarce. Hence, the objective of this investigation was to evaluate in what manner phototherapy using red light influences the growth and arrangement of C. albicans biofilm. For the twice-daily treatment, we adapted our laboratory&#39;s previous protocols9,12 to provide an easy and reproducible biofilm model that delivers answers regarding viability, dry-weight and extracellular polysaccharides amounts after red light treatment. The same protocol can be used for testing other therapies.

<table border="0" cellpadding="0" cellspacing="0" style="width:1115px;" width="1115">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Clorhexidine 20%&#160;</td>
			<td style="width:372px;">Sigma-Aldrich</td>
			<td style="width:123px;">C9394</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dextrose (D-Glucose) Anhydroous</td>
			<td style="width:372px;">Fisher Chemical</td>
			<td style="width:123px;">D16-500</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 200 proof</td>
			<td style="width:372px;">Decon Laboratories</td>
			<td style="width:123px;">DSP-MD.43</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LumaCare LC-122 A&#160;</td>
			<td style="width:372px;">LumaCare Medical Group, Newport Beach, CA, USA&#160;</td>
			<td style="width:123px;"></td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">NaCl&#160;</td>
			<td style="width:372px;">Fisher Chemical</td>
			<td style="width:123px;">S641-500</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH&#160;</td>
			<td style="width:372px;">Fisher Bioreagents&#160;</td>
			<td style="width:123px;">BP 359-500</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol 5%</td>
			<td style="width:372px;">Milipore Sigma</td>
			<td style="width:123px;">843984</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">RPMI 1640 buffered with 3-(N-morpholino)</td>
			<td style="width:372px;">Sigma</td>
			<td style="width:123px;">R7755</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Sabouraud dextrose agar supplemented with chloramphenicol</td>
			<td style="width:372px;">Acumedia</td>
			<td style="width:123px;">7306A</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid&#160;</td>
			<td style="width:372px;">Fisher Chemical</td>
			<td style="width:123px;">SA200-1</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast nitrogen base&#160;</td>
			<td style="width:372px;">Difco</td>
			<td style="width:123px;">DF0392-15-9</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-(N-morpholino)propanesulfonic acid MOPS</td>
			<td style="width:372px;">Sigma-Aldrich</td>
			<td style="width:123px;">M1254</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">&#160;24-well polystyrene plate&#160;</td>
			<td style="width:372px;">Falcon</td>
			<td style="width:123px;">353935</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

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.

Figure 2&#160;displays the outcomes of Log10 CFU/mL of C. albicans after per diem treatments with red light for 1 min. Red light significantly reduced the Log10 CFU/mL compared to the NC (p = 0.004). Figure 3 presents the outcomes of the biomass (mg) of C. albicans biofilms after daily treatments. All treated groups showed reduction of the biomass compared to the NC (p = 0.000) and the red ...

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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.

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 ...

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Research

JoVE Journal

Immunology and Infection

7.6K Views.  Indiana University School of Dentistry. 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·cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Video: Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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 ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

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. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

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 ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

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 ...

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.

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.

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

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, ...