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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M., Wijesinghe, G. K., Jayarathna, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+media+profoundly+affect+Candida+albicans+and+Candida+tropicalis+growth,+adhesion+and+biofilm+development.">Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development.</a> Memórias Do Instituto Oswaldo Cruz. 111 (11), 697-702 (2016).</li><li>Kadosh, D., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+Candida+albicans+filamentous+growth+program+by+relief+of+transcriptional+repression:+a+genome-wide+analysis.">Induction of the Candida albicans filamentous growth program by relief of transcriptional repression: a genome-wide analysis.</a> Molecular biology of the cell. 16 (6), 2903-2912 (2005).</li><li>Paschoal, M. 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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 colonies that are more than seven days old, not to store plates at 4&#176;C, and not to re-streak cells from existing plates. Likewise, strains should be used prior to 18 hours of overnight growth13.
A study limitation is that it was performed in vitro. Whilst in vitro studies have greatly increased the understanding of the biology of biofilm, they do not precisely represent in vivo conditions14. However, in vitro tests provide high-throughput screening in addition to being a cheap and simple methodology14&#160;to answer questions regarding new antibiofilm therapies. Choosing the correct culture media for each phase of biofilm development is an important step for the success of the method. RPMI 1640 is a nutrient-rich medium that has amino acids and simulates the composition of human fluids15. RPMI 1640 contains L-glutamine, L-arginine, L- asparagine in addition to vitamins and inorganic salts. Nevertheless, YNB media has an elevated amount of glucose (18 g/L) compared to RPMI 1640 medium (2 g/L glucose). The high glucose content has been described to increase the planktonic growth of Candida species16. In contrast, the existence of higher concentrations of amino acids will help biofilm growth in RPMI medium compared to YNB medium16. Qualitative data applying SEM micrographs showed that the structural design of C. albicans biofilms in RPMI presents a complex organization with a solid growth of yeasts with ramifying hyphae, budding elements and bud scars with an abundant extracellular matrix16. Such outcomes are in accordance with a previous study that reported that RPMI 1640 augmented the hyphal formation in C. albicans biofilms15. These results demonstrate the differences in substrate utilization by Candida throughout different biofilm growth phases and show the importance of changing media during planktonic growth and biofilm formation.
The selection of the correct temperature and pH to cultivate C. albicans biofilms is also important to accomplish the method with the adequate formation of hyphae. C. albicans undertakes the conversion from blastospores to filaments in reaction to conditions that mimic the milieu of mammalian host tissues17. These conditions involve growing at body temperature (37 &#176;C) and at neutral pH17, and this is the reason why the pH of the RPMI medium is adjusted to 7.
The methods applied in this study are significant since biofilms of C. albicans SN 425 were characterized before12, showing to have great amounts of EPS-soluble and -insoluble, which makes it a reliable method to analyze the outcome of the lights on the extracellular polysaccharides. Moreover, the same non-coherent light device applied in the experiments was successfully applied to bacterial planktonic suspensions18&#160;and biofilms9, and the utmost benefit of this device is the reduction in treatment time, what makes the device more feasible for clinical applications18. The analysis of daily phototherapy applied to C. albicans biofilms is significant since the methodology applied in the present study simulates a treatment that is fast and can be easily done by the patient at home.
Red light daily treatment meaningfully reduced C. albicans viable colony count and biofilm biomasses. More studies might try a combination of treatments, starting with phototherapy and later applying topical antifungal. This strategy might assist in disorganizing the extracellular matrix shielding C. albicans biofilm, permitting better drug infiltration through the biofilm to reach and finally eradicate C. albicans cells. Considering the limitations of this in vitro experiment, the use of red light for 1 min may assist as an adjuvant to topic antifungals on the treatment of oral candidiasis.

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><tbody><tr><td>Clorhexidine 20%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C9394</td><td></td></tr><tr><td>Dextrose (D-Glucose) Anhydroous</td><td>Fisher Chemical</td><td>D16-500</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon Laboratories</td><td>DSP-MD.43</td><td></td></tr><tr><td>LumaCare LC-122 A&amp;nbsp;</td><td>LumaCare Medical Group, Newport Beach, CA, USA&amp;nbsp;</td><td></td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Fisher Chemical</td><td>S641-500</td><td></td></tr><tr><td>NaOH&amp;nbsp;</td><td>Fisher Bioreagents&amp;nbsp;</td><td>BP 359-500</td><td></td></tr><tr><td>Phenol 5%</td><td>Milipore Sigma</td><td>843984</td><td></td></tr><tr><td>RPMI 1640 buffered with 3-(N-morpholino)</td><td>Sigma</td><td>R7755</td><td></td></tr><tr><td>Sabouraud dextrose agar supplemented with chloramphenicol</td><td>Acumedia</td><td>7306A</td><td></td></tr><tr><td>Sulfuric acid&amp;nbsp;</td><td>Fisher Chemical</td><td>SA200-1</td><td></td></tr><tr><td>Yeast nitrogen base&amp;nbsp;</td><td>Difco</td><td>DF0392-15-9</td><td></td></tr><tr><td>3-(N-morpholino)propanesulfonic acid MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>&amp;nbsp;24-well polystyrene plate&amp;nbsp;</td><td>Falcon</td><td>353935</td><td></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 light treated groups presented similar reduction of biomass to that observed in the PC. Figure 4 and Figure 5 display inferior amounts of C. albicans EPS-soluble and EPS-insoluble in PC compared to NC (p = 0.000). Even though not statistically significant, per diem application of red light for 1 min to C. albicans biofilms numerically diminished the amounts of EPS-soluble and EPS- insoluble.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59326/59326fig1.jpg" /> 
Figure 1. Growth curve of C. albicans strain SN 425. Planktonic culture was made in YNB medium supplemented with 100 mM of glucose and incubated at 37 &#176;C. The optical density (OD at 540 nm) and Log10 CFU/mL were determined over time. Standard deviation is shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59326/59326fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59326/59326fig2.jpg" /> 
Figure 2. Mean and standard deviations of Log10 CFU/mL of C. albicans. Assessments were made between the treatment with red light twice a day and the controls-0.12% CHX (PC) and 0.89% NaCl (NC). Equal letters represent statistical similarity between groups (p &#62; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59326/59326fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59326/59326fig3.jpg" /> 
Figure 3. Mean and standard deviations of dry-weight (mg) of C. albicans. Assessments were made between the treatment with red light twice a day and the controls-0.12% CHX (PC) and 0.89% NaCl (NC). Equal letters represent statistical similarity between groups (p &#62; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59326/59326fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59326/59326fig4.jpg" /> 
Figure 4. Mean and standard deviations of EPS-soluble amount in C. albicans biofilm (&#181;g/mg of dry-weight). Comparisons were made between the treatment with red light twice a day and the controls-0.12% CHX (PC) and 0.89% NaCl (NC). Equal letters represent statistical similarity between groups (p &#60; 0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59326/59326fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59326/59326fig5.jpg" /> 
Figure 5. Mean and standard deviations of EPS-insoluble content in C. albicans biofilm (&#181;g/mg of dry-weight). Assessments were made between the treatment with red light twice a day and the controls-0.12% CHX (PC) and 0.89% NaCl (NC). Equal letters represent statistical similarity between groups (p &#60; 0.001).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59326/59326fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth at JoVE.com

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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Nanyang Technological University

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JoVE Journal

Immunology and Infection

7.7K 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 ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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. ...

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, ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Analyzing Petite Colony Formation and Drug Resistance in Candida glabrata

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Author Spotlight: Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug&#45;Resistant Candida for Antifungal Research

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

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

Microtiter Dish Biofilm Formation Assay

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

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

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

Clarifying and Imaging Candida albicans Biofilms

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy