Name: A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms (Video) | JoVE Journal
Uploaded: 2010-10-21T00:00:00.000+00:00
Duration: 7 min 19 s
Description: Learn a 96-well microtiter plate method for forming Candida albicans biofilms and testing antifungal susceptibility. This reproducible technique enables high-throughput screening of antifungal agents.

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5 minutes

Biofilm-related work in the laboratory is funded by grants numbered R21DE017294 and R21AI080930 from the National Institute of Dental &amp; Craniofacial Research and the National Institute for Allergy and Infectious Diseases (to J.L.L.-R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDCR, the NIAID or the NIH.

1. Preparation of C. albicans
C. albicans is a Risk Group 1/BSL1 microorganism. Always remember to use good aseptic/sterile techniques for work with this microorganism and follow institutional procedures for proper disposal of biohazard materials.
<ol>
	<li>Prepare an overnight culture of C. albicans in YPD (Yeast Peptone Dextrose) liquid medium by inoculating a single colony of C. albicans into 25 mL of YPD.</li>
	<li>Incubate culture in an orbital shaker (about 180 rpm) at 30 °C overnight. Most C. albicans strains will grow as yeast cells under these conditions.</li>
	<li>Centrifuge the overnight cultures (about 3,000 rpm, 5-10 minutes), wash cells twice with sterile PBS, and resuspend the final pellet in about 20-25 mL of RPMI 1640 medium buffered with 165 mM morpholinepropanesulfonic acid to pH 7.0 and pre-warmed at 37 °C (from now on this medium is referred to as simply "RPMI 1640"). </li>
	<li>Count cells using an hemacytometer. After counting, prepare a suspension of cells at a final density of 1.0 x 106 cells/ mL in RPMI 1640. Note: Because cells have a tendency to aggregate, it is important to vortex vigorously between washings and before pipetting.</li>
</ol>
2. Setting Up the 96-well Microtiter Plate for the Formation of the Biofilm
<ol>
	<li> Using a multichannel pipette add 100 μL of C. albicans suspension into selected wells of the 96-well microtiter plate(-s). Do not add cells to wells in column 12, as these will serve as negative controls.</li>
	<li>Cover the entire microtiter plate with its original lid, seal with parafilm, place inside an incubator and incubate for 24 h at 37 °C. The length of incubation can be adapted to the specific experimental design. For example, it is possible to examine the kinetics of biofilm formation over a period of 24 - 48 hours by seeding multiple plates and processing each plate at different time points (i.e. 2, 4, 8, 12, 24 and 48 h)</li>
	<li>After biofilm formation, using a multichannel pipette aspirate the medium carefully as not to touch and disrupt the biofilms that have formed in each of the wells. </li>
	<li>Using a multichannel pipette wash plates three times in sterile PBS (200 μL per well). Alternatively, use an automated microtiter plate washer. Between washes, and particularly after the last wash, drain the plates in an inverted position by blotting with paper towels to remove any residual PBS. At this point biofilms formed on the bottom of the wells should be clearly visible even by the naked eye and can also be visualized using an inverted microscope (Figure 1). Biofilms are now ready to be processed for antifungal susceptibility testing assays. (If the main purpose of the experiment is to assess the extent of biofilm formation, the plates are ready to be processed using the colorimetric method. For this, the XTT/menadione reagent (see Step 3 below) can be added and the resulting color read using a microtiter plate reader).</li>
</ol>
3. Antifungal Susceptibility Testing of Biofilms
<ol>
	<li> From stock solutions or powder, prepare a working solution in RPMI 1640 medium of each antifungal to be tested. Typical high concentrations are 1,024 μg/ mL for fluconazole, and 16 μg/ mL for both amphotericin B and caspofungin. Other concentrations may be used for different agents.</li>
	<li>Using a multichannel pipette, add 200 μL of the high working concentration of antifungal to the corresponding wells in column 1 of each microtiter plate containing fungal biofilms. </li>
	<li>Add 100 μL of RPMI 1640 to each well in columns 2 to 10. </li>
	<li>Add 100 μL of RPMI 1640 to wells in column 11. </li>
	<li>Remove 100 μL of antifungal agent from the wells of column 1 and add to the adjacent wells in column 2 (already containing 100 μL of medium). </li>
	<li>Mix the contents well by pipetting up and down to perform a serial doubling dilution, and remove the tips. </li>
	<li>Repeat moving right until the wells of column 10, after which the final 100 μL volume from the wells of column 10 after mixing is discarded. In this way, a series of doubling dilutions of your agent(-s) of interest have been created; from most concentrated in wells of column 1 to least concentrated in wells of column 10. Unchallenged biofilms in column 11 will serve as positive controls, and empty wells in column 12 will serve as negative controls. </li>
	<li>Cover the plates with their lids, seal with parafilm and incubate for 24 -48 h at 37 °C.</li>
	<li>After the incubation period wash the plates as in Step 2.4 before (3 x PBS).</li>
	<li>Using a multichannel pipette add 100 μL of XTT/menadione solution to each well containing a pre-washed biofilm as well as to negative control wells for the measurement of background XTT-colorimetric levels.
	<ol>
		<li>The XTT is prepared as a saturated solution at 0.5 g/L in sterile Ringer's lactate, PBS or saline, which needs to be filter-sterilized using a 0.22 μm-pore size filter. The XTT solution is light sensitive, so it should be covered with aluminum foil during preparation. Once prepared and filter-sterilized, aliquot into 10 mL working volumes, and store at -70 °C. Protect the tubes from light using aluminum foil. Thaw only as many tubes as needed for a particular experiment just prior to use.</li>
		<li>Menadione is prepared as a 10 mM stock solution in 100% acetone, aliquoted into smaller volumes (about 50 μL) and stored at -70°C. Prepare the XTT/menadione solution just prior to use, by adding 1 μL of the stock solution of menadione to a tube containing 10 mL of the thawed XTT solution.</li>
	</ol>
 </li>
	<li>Cover the plates in aluminum foil and incubate in the dark for 2 h at 37 °C. </li>
	<li>Uncover the plates. Using a multichannel pipette remove 80 μL of the resulting colored supernatant from each well and transfer into the corresponding wells of a new microtiter plate. </li>
	<li>Read the plate(-s) in a microtiter plate reader at 490 nm. </li>
	<li>Calculate the sessile minimum inhibitory concentrations SMIC50 and SMIC80, which are the antifungal concentrations at which a 50% or 80% decrease in colorimetric readings are detected in comparison to the control biofilms formed in the absence of antifungal drug (in this case values for column 11, remember also to subtract values from negative controls from wells in column 12 containing XTT only). SMIC results can be presented as a Table (i.e. multiple isolates against multiple antifungals) or alternatively, results for each individual fungal isolate against each antifungal can be presented as a graph by plotting percent inhibition versus antifungal concentration.</li>
</ol>
Example: After subtracting the values in the negative control, the average O.D. of control biofilms formed in column 11 is 1.32. The SMIC50 is the lowest antifungal concentration leading to &gt; 50% reduction in colorimetric readings, in this case less than 1.32 x 50/100 = 0.66. Likewise, the SMIC80 is the lowest antifungal concentration leading to &gt; 80% reduction in colorimetric readings, in this case less than 1.32 x 20/100 = 0.264.
4. Representative Results
Figure 1 shows a microphotograph of a C. albicans biofilm formed on the bottom of a well in a 96 well microtiter plate taken using an inverted microscope. Figure 2 shows XTT-colorimetric readings (OD490 values) for each of 11 biofilms of a C. albicans wild type strain formed in each of the 8 different rows of the same 96 well microtiter plate. Figure 3 shows the activity of amphotericin B at different concentrations against C. albicans biofilms; arrows indicate SMIC50 and SMIC80 values.
<img src="/files/ftp_upload/2287/2287fig1.jpg" alt="Bacterial growth comparison; microscope and SEM images; cell morphology study; scale bars visible." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1.(A) Panel A shows a microphotograph taken using a camera attached to an inverted microscope of a C. albicans biofilm formed on the bottom of the well after aspiration of RPMI medium and subsequent washings with PBS. (B) A micrograph of a typical C. albicans biofilm visualized using scanning electron microscopy. Bars are 100 μm and 10 μm for panels A and B respectively.
<img src="/files/ftp_upload/2287/2287fig2.jpg" alt="XTT assay results; colorimetric analysis graph displaying sample readings, comparative data." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Formation of multiple equivalent C. albicans biofilms in 96-well microtiter plates. Colorimetric readings (OD490 values) from XTT-reduction assays of biofilms formed by a C. albicans wild type in wells of microtiter plates. Values are for 11 independent biofilms formed in each of 8 different rows of the same 96 well microtiter plate. Results for the different rows were compared by one-way analysis of variance and using the Bartlett's test for homogeneity of variances and the Bonferroni's multiple comparison post-test. No statistically significant differences were noted when comparing all pairs of rows to each other (P &gt; 0.05).
<img src="/files/ftp_upload/2287/2287fig3.jpg" alt="Antifungal susceptibility graph, XTT assay, Amphotericin B dose-response, SMIC50/80 values." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Typical results of antifungal susceptibility testing against C. albicans biofilms. Graph depicting typical results of the efficacy of different amphotericin B concentrations against biofilms of a C. albicans wild type strain. Values are expressed as average percent colorimetric readings for XTT-reduction assays compared to control wells. SMIC50 and SMIC80 values are indicated by arrows.

<ol><li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+activity+of+caspofungin+against+Candida+albicans+biofilms%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">In vitro activity of caspofungin against Candida albicans biofilms.</a> Antimicrob Agents Chemother. 46, 3591-3596 (2002).</li>
<li>Hawser, S. P., Norris, H., Jessup, C. J., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+a+2%2C3-bis%282-methoxy-4-nitro-5-sulfophenyl%29-5-%5B%28phenylamino%29carbonyl%5D-2H-t+etrazolium+hydroxide+%28XTT%29+colorimetric+method+with+the+standardized+National+Committee+for+Clinical+Laboratory+Standards+method+of+testing+clinical+yeast+isolates+for+susceptibility+to+antifungal+agents%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t etrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.</a> J Clin Microbiol. 36, 1450-1452 (1998).</li>
<li>Kuhn, D. M., Balkis, M., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uses+and+limitations+of+the+XTT+assay+in+studies+of+Candida+growth+and+metabolism%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Uses and limitations of the XTT assay in studies of Candida growth and metabolism.</a> J Clin Microbiol. 41, 506-508 (2003).</li>
<li>Kuhn, D. M., George, T., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antifungal+susceptibility+of+Candida+biofilms%3A+Unique+efficacy+of+amphotericin+B+lipid+formulations+and+echinocandins%5BTitle%5D)+AND+%22Antimicrob.+Agents+Chemother%22%5BJournal%5D)">Antifungal susceptibility of Candida biofilms: Unique efficacy of amphotericin B lipid formulations and echinocandins.</a> Antimicrob. Agents Chemother. 46, 1773-1780 (2002).</li>
<li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C.+albicans+transcription+factor+Bcr1p%3A+a+regulator+of+cell-surface+genes+and+biofilm+formation%5BTitle%5D)+AND+%22Current+Biol%22%5BJournal%5D)">C. albicans transcription factor Bcr1p: a regulator of cell-surface genes and biofilm formation.</a> Current Biol. 15, 1150-1155 (2005).</li>
<li>Ramage, G., Saville, S. P., Thomas, D. P., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+biofilms%3A+an+update%5BTitle%5D)+AND+%22Eukaryot+Cell%22%5BJournal%5D)">Candida biofilms: an update.</a> Eukaryot Cell. 4, 633-638 (2005).</li>
<li>Ramage, G., Vande Walle, K., Wickes, B. L., p, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob Agents Chemother. 45, 2475-2479 (2001).</li>
<li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antifungal+combinations+against+Candida+albicans+biofilms+in+vitro%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Antifungal combinations against Candida albicans biofilms in vitro.</a> Antimicrob Agents Chemother. 47, 3657-3659 (2003).</li>
<li>Ramage, G., VandeWalle, K., Bachmann, S. P., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+pharmacodynamic+properties+of+three+antifungal+agents+against+preformed+Candida+albicans+biofilms+determined+by+time-kill+studies%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">In vitro pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies.</a> Antimicrob Agents Chemother. 46, 3634-3636 (2002).</li>
<li>Ramage, G., VandeWalle, K., Wickes, B. L., López-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characteristics+of+biofilm+formation+by+Candida+albicans%5BTitle%5D)+AND+%22Rev.+Iberoam.+Micol%22%5BJournal%5D)">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li>
<li>Tellier, R., Krajden, M., Grigoriew, G. A., Campbell, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innovative+endpoint+determination+system+for+antifungal+susceptibility+testing+of+yeasts%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Innovative endpoint determination system for antifungal susceptibility testing of yeasts.</a> Antimicrob Agents Chemother. 36, 1619-1625 (1992).</li>
<li>Nett, J., Andes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+albicans+biofilm+development%2C+modeling+a+host-pathogen+interaction%5BTitle%5D)+AND+%22Curr+Opin+Microbiol%22%5BJournal%5D)">Candida albicans biofilm development, modeling a host-pathogen interaction.</a> Curr Opin Microbiol. 9, 340-345 (2006).</li>
</ol>

JLL-R owns equity in MicrobeHTS Technologies, Inc., which is developing antifungal agents. MicrobeHTS Technologies, Inc. provided no financial support for these studies.

Here we describe a simple, rapid, economical and highly reproducible 96 well microtiter plate model for the formation of Candida biofilms coupled with a colorimetric method that measures the metabolic activities of cells within the biofilm using XTT. This 96 well microtiter plate model for biofilm formation was originally developed for C. albicans but can be used for other Candida spp. and easily adapted for other fungal organisms. The method can be used to examine multiple parameters and facto...

<table><tbody><tr><td>Sabouraud-dextrose agar</td><td>BD Biosciences</td><td>211584</td><td>To prepare plates for fresh subcultures of fungal isolates</td></tr><tr><td>YPD: Yeast peptone dextrose</td><td>US Biological</td><td>Y2076</td><td>Medium for propagation of overnight liquid cultures</td></tr><tr><td>RPMI-1640 without sodium bicarbonate supplemented with L-glutamine</td><td>Cellgro</td><td>50-020-PB</td><td>Liquid medium for biofilm formation</td></tr><tr><td>Morpholinepropanesulfonic acid (MOPS)</td><td>Fisher Scientific</td><td>BP308</td><td>To buffer RPMI 1640</td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Sigma-Aldrich</td><td>P4417</td><td>Buffer for washes</td></tr><tr><td>XTT sodium salt</td><td>Sigma-Aldrich</td><td>X4251</td><td>See above for preparation instructions</td></tr><tr><td>Ringer&amp;rsquo;s lactate</td><td>Hospira Inc.</td><td>NDC0409-7953-09</td><td>For preparation of XTT solution</td></tr><tr><td>Menadione</td><td>Sigma-Aldrich</td><td>M5625</td><td>Caution: hazardous by skin contact, inhalation or ingestion</td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>08-757-12</td><td></td></tr><tr><td>15 ml conical centrifuge tubes</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Multichannel pipette and tips</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Incubator</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Microtiter plate reader</td><td>Any Supplier</td><td></td><td></td></tr></tbody></table>

a 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilms

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.

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 ...

The overall goal of this procedure is to form candida albicans biofilms, and examine their susceptibility against antifungal agents. This is accomplished by first forming these fungal biofilms on the wells of 96. Well microtiter plates then antifungals are added to the preformed biofilms and incubated for 24 to 48 hours following this incubation.A chole metric method that measures metabolic activity of cells within the biofilms is used to measure the activity of these antifungals in order to establish the minimum inhibitory concentration of antifungals against biofilms. Ultimately, results can be obtained that show the ability to form biofilms and antifungal susceptibility testing through a simple and easy 96 well microtiter plate based method coupled to a color metric reading. The main advantage of this technique is that it is easy, relatively inexpensive, highly reproducible, and allows for the formation of multiple equivalent biofilms.In addition, it is compatible with the 96 well platform technology commonly available in most research and clinical laboratories. The implications of this technique extend towards the therapy of canids. Since very often, these infections are associated with the biofilm formation.Using proper microbiological handling techniques, retrieve a loop full of cells from a fresh subculture of albicans on a SAU dextrose copperplate Alan and inoculate into a 125 milliliter erlenmeyer flask containing 25 milliliters of yeast pepane dextrose broth placed the culture in an orbital shaker set at 180 RRP M and 30 degrees Celsius, and incubated overnight following the overnight incubation. Pour the yeast cultures into 50 milliliter conical tubes and centrifuge at 3000 RBM for five to 10 minutes. Discard the supernatant vortex.The pellet vigorously to desegregate the cells. Then add sterile PBS to wash before centrifuging the cultures again. Repeat this washing step one more time than re bend the cells.In about 20 milliliters of RPMI 1640, medium buffered to pH 7.0. Using a solution of 165 millimolar mops and pre warmed at 37 degrees Celsius from this suspension, prepare a one to 100 dilution using a standard hemo cytometer and a bright field microscope. Count the cells.Then prepare a final suspension of cells at one times 10 of the six per milliliter in warm buffered RPMI 1640 use. Using a multi-channel pipette at 100 microliters of sea albican suspension into selected wells of a sterile 96 well flat bottom microtiter plate. Leave the wells in column 12 empty as they will serve as negative controls.After covering and sealing the plate with paraform, incubate it at 37 degrees Celsius for 24 hours to allow the biofilms to form. Following the incubation, gently aspirate and discard the media from each well. Taking care not to disrupt the biofilms formed at the bottom.To wash at 200 microliters of PBS to each well and drain by carefully inverting and blotting the plate on a stack of paper towels. Repeat this wash step three times. At this point, biofilms are detectable by eye and can be visualized using an inverted microscope using SEM.We can appreciate that the biofilms consist of an intertwined mixture of filaments and yeast cells. Once biofilm formation has been verified, testing of antifungal agents can commence using buffered RPMI 1640 medium prepare high concentration working solutions of the antifungal agents to be tested. Typical high concentrations are 1024 micrograms per milliliter for fluconazole and 16 micrograms per milliliter for amphotericin B and CASPO fungi beri.Next, add 200 microliters of antifungal solutions to wells in column one and 100 microliters of buffered RPMI 1640. Medium to wells in columns two through 11. Perform a series of serial doubling dilutions by transferring 100 microliters of the high concentration antifungal solutions from the wells of column one into those of column two.After gently pipetting up and down to mix pipette 100 microliters from wells of column two into those of column three and so forth until column 10. Do not add any antifungal solution to column 11, as it will serve as a positive control and discard the last 100 microliters from column 10 wells. All wells, except those in column 12.Should now have a final volume of 100 microliters. Cover the plate, seal it with parfum and incubate it at 37 degrees Celsius for 24 to 48 hours to allow the antifungals to act following the incubation. And after washing the plate three times in PBS as previously described, add 100 microliters of XTT medion solution per well to all wells, including the negative control wells.In column 12. Cover the plate, wrap it in aluminum foil to protect it from the light and incubate it at 37 degrees Celsius for two hours. To allow color development following incubation, unwrap the plate.The initially clear XTT solution will have turned orange after being processed by metabolically active cells. Transfer 80 microliters of the colored supernatants to a fresh, flat bottom microtiter plate. Use a colormetric microtiter plate reader set at a wavelength of 490 nanometers to read the plate as shown here In the absence of antifungals, colormetric readings were untreated.SEA applicant's biofilms in different rows are equivalent. In order to evaluate the effect of the antifungal compounds tested first, subtract an average background value obtained from the negative control wells in column 12 from all the other values. Next, calculate the percent inhibition using the positive controls in row 11.As the maximal values, the antifungal concentrations at which a 50%or 80%decrease in colormetric readings is detected, are known as the Cecil minimum inhibitory concentrations. These values are typically used as a reference measure of the potency of a compound. Using this simple high throughput method, fungal biofilms can be formed in vitro in 24 hours, and selected compounds can be screened for antifungal activity After its initial development.This technique paved the way for the researchers in the field of medical mycology to explore the biofilm formation and to examine the activity of the antifungal agents. Again, the cells in biofilms.

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Research

JoVE Journal

Immunology and Infection

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Microtiter Dish Biofilm Formation Assay

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

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

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

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

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

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

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.

Clarifying and Imaging Candida albicans Biofilms

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

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.

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

Learn a 96-well microtiter plate method for forming Candida albicans biofilms and testing antifungal susceptibility. This reproducible technique enables high-throughput screening of antifungal agents.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms (Video) | JoVE Journal

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

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