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 orbita...

<ol>
	<li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activity+of+caspofungin+against+Candida+albicans+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+and+limitations+of+the+XTT+assay+in+studies+of+Candida+growth+and+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+susceptibility+of+Candida+biofilms:+Unique+efficacy+of+amphotericin+B+lipid+formulations+and+echinocandins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+albicans+transcription+factor+Bcr1p:+a+regulator+of+cell-surface+genes+and+biofilm+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+biofilms:+an+update.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+combinations+against+Candida+albicans+biofilms+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+pharmacodynamic+properties+of+three+antifungal+agents+against+preformed+Candida+albicans+biofilms+determined+by+time-kill+studies.">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., L&#243;pez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+endpoint+determination+system+for+antifungal+susceptibility+testing+of+yeasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilm+development,+modeling+a+host-pathogen+interaction.">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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sabouraud-dextrose agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211584</td>
<td>To prepare plates for fresh subcultures of fungal isolates</td>
</tr>
<tr>
<td>YPD: Yeast peptone dextrose</td>
<td>&nbsp;</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>&nbsp;</td>
<td>Cellgro</td>
<td>50-020-PB</td>
<td>Liquid medium for biofilm formation</td>
</tr>
<tr>
<td>Morpholinepropanesulfonic acid (MOPS)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP308</td>
<td>To buffer RPMI 1640</td>
</tr>
<tr>
<td>Phosphate buffered saline, PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>Buffer for washes</td>
</tr>
<tr>
<td>XTT sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>X4251</td>
<td>See above for preparation instructions</td>
</tr>
<tr>
<td>Ringer's lactate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC0409-7953-09</td>
<td>For preparation of XTT solution</td>
</tr>
<tr>
<td>Menadione</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5625</td>
<td>Caution: hazardous by skin contact, inhalation or ingestion</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430790</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3595</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette and tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microtiter plate reader</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms at JoVE.com

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.

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 ...

a-96-well-microtiter-plate-based-method-for-monitoring-formation

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JoVE Journal

Immunology and Infection

44.4K Views.  University of Texas San Antonio - UTSA. 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.