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 &deg;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 &deg;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 &mu;L 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 &deg;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 &mu;L 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 &mu;g/ mL for fluconazole, and 16 &mu;g/ mL for both amphotericin B and caspofungin. Other concentrations may be used for different agents.</li>
	<li>Using a multichannel pipette, add 200 &mu;L 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 &mu;L of RPMI 1640 to each well in columns 2 to 10. </li>
	<li>Add 100 &mu;L of RPMI 1640 to wells in column 11. </li>
	<li>Remove 100 &mu;L of antifungal agent from the wells of column 1 and add to the adjacent wells in column 2 (already containing 100 &mu;L 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 &mu;L 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 &deg;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 &mu;L 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 &mu;m-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 &deg;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 &mu;L) and stored at -70&deg;C. Prepare the XTT/menadione solution just prior to use, by adding 1 &mu;L 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 &deg;C. </li>
	<li>Uncover the plates. Using a multichannel pipette remove 80 &mu;L 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="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 &mu;m and 10 &mu;m for panels A and B respectively.
<img src="/files/ftp_upload/2287/2287fig2.jpg" 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="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/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 ...

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Immunology and Infection

44.7K Views.  University of Texas San Antonio - UTSA. Descriviamo un metodo semplice, rapido e robusto per la formazione di Candida albicans Biofilm su 96 micropiastre bene e la sua utilità nei test di suscettibilità antifungina di cellule all'interno di biofilm.

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

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 ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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 ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

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, ...