The authors thank professor Paul Cos and LMPH, University of Antwerp, Belgium for his support during the filming process in his laboratory. This work was funded by Academy of Finland projects (projects 272266 and 282981) and Svenska Tekniska Vetenskapsakademien i Finland. The technical contributions of MSc Janni Kujala are acknowledged.

1. Growing the Bacteria
<ol>
	<li>Pre-culture the bacteria overnight in a tryptic soy broth (TSB) at 37 &#176;C with 220 rpm shaking (16-18 hr).</li>
	<li>Dilute the pre-culture 100-1,000 times (depending on the growth rate of the bacteria; here, 1,000 times is used for S. aureus) in fresh TSB and let it grow at 37 &#176;C and 200 rpm to reach exponential growth (optical density at 595 nm (OD595) between 0.2 and 0.6). 
	NOTE: This step requires strain-specific optimization.</li>
</ol>
2. Biofilm Formation: Pre- and Post-exposure
<ol>
	<li>Dilute the exponentially grown culture 100 times (this equals approximately 106 colony-forming units per milliliter (CFU/ml)).</li>
	<li>Pre-exposure protocol
	<ol>
		<li>For untreated control samples, add 200 &#181;l of the diluted bacterial culture per well of a sterile 96-microwell plate.</li>
		<li>Add 4 &#181;l of a test compound or a control antibiotic (50x stock solution) and 196 &#181;l of the diluted bacterial culture per well.</li>
		<li>Incubate it at 37 &#176;C on a plate shaker at 200 rpm for 18 hr. 
		Note: Here we use a shaker with a 2 mm orbit.</li>
	</ol>
	</li>
	<li>Post-exposure protocol
	<ol>
		<li>For all samples, add 200 &#181;l of the diluted bacterial culture per well of a sterile 96-microwell plate.</li>
		<li>Incubate it at 37 &#176;C on a plate shaker at 200 rpm for 18 hr. 
		Note: Here we use a shaker with a 2 mm orbit.</li>
		<li>Remove the entire planktonic solution carefully, without touching the biofilm, using a multichannel pipette.</li>
		<li>Add 4 &#181;l of a test compound or a control antibiotic (50x stock solution) and 196 &#181;l of TSB per well.</li>
		<li>Incubate it at 37 &#176;C on a plate shaker at 200 rpm for an additional 24 hr. 
		Note: Here we use a shaker with a 2 mm orbit.</li>
	</ol>
	</li>
</ol>
3. Resazurin Staining Protocol for the Viability Assessment of Biofilms
<ol>
	<li>Prepare a stock solution of 0.1 mg/ml resazurin (0.4 mM) in sterile PBS. Keep this stock sterile, protected from light exposure, and at 4 &#176;C.</li>
	<li>Dilute the resazurin stock 1:50 in sterile phosphate-buffered saline (PBS) to achieve a final concentration of 20 &#181;M.</li>
	<li>Transfer the entire planktonic solution (leaving the biofilms in the wells) carefully, without touching the biofilms or creating air bubbles, to a separate, clean 96-well plate using a multichannel pipette.</li>
	<li>Wash the biofilms once with sterile PBS by adding 200 &#181;l per well, and remove it carefully using a multichannel pipette.</li>
	<li>Add 200 &#181;l of the diluted resazurin per well of the biofilm plate using a multichannel pipette.</li>
	<li>Incubate it in darkness, at room temperature (RT), and 200 rpm, shaking for approximately 20 min until the untreated biofilm controls are evenly pink.</li>
	<li>Measure the fluorescence at &#955;exc = 560 nm and &#955;em = 590 nm with a plate reader (top reading).</li>
</ol>
4. Crystal Violet Staining Protocol for Biomass Quantification of Biofilms Using the Same Plate as in Step 3
<ol>
	<li>Remove the resazurin stain carefully from the wells using a multichannel pipette.</li>
	<li>Fix the biofilms with 200 &#181;l of methanol for 15 min.</li>
	<li>Let the plate air dry for 10 min.</li>
	<li>Add 190 &#181;l of 0.02 % (vol/vol, diluted in deionized water) crystal violet solution, carefully using a multichannel pipette. Avoid touching the sides of the wells with the stain while pipetting and prevent the formation of air bubbles by not pressing the pipette to complete blow-out.</li>
	<li>Incubate it for 5 min at RT.</li>
	<li>Remove the stain carefully, using a multichannel pipette.</li>
	<li>Wash it twice with deionized water (200 &#181;l each time).</li>
	<li>Let the wells dry for 5 min at RT and dissolve the remaining stain in 96% ethanol or 33% acetic acid.</li>
	<li>Incubate it for 1 hr at RT and read the absorbance at 595 nm.</li>
</ol>
5. Evaluating the Bactericidal Effect on Planktonic Bacteria Using the Same Sample Plate as for the Biofilms
<ol>
	<li>Measure the turbidity at 595 nm of the plate with the planktonic solution from 3.3.</li>
	<li>Stain the planktonic bacteria with resazurin by adding 10 &#181;l of the resazurin stock per well. Mix well by pipetting.</li>
	<li>Incubate it in darkness at RT for approximately 5 min, until the untreated controls are evenly pink.</li>
	<li>Measure the fluorescence at &#955;exc = 560 nm and &#955;em = 590 nm.</li>
</ol>
6. Wheat Germ Agglutinin Staining for Matrix Quantification and Fluorescence Microscopy Imaging of Biofilms
<ol>
	<li>Matrix quantification
	<ol>
		<li>Prepare a 5 &#181;g/ml solution of the WGA probe in sterile PBS.</li>
		<li>Use a parallel sample plate for this staining. Remove the planktonic solution from the wells and wash once with sterile PBS (200 &#181;l per well), carefully using a multichannel pipette without touching the biofilms.</li>
		<li>Add 200 &#181;l of WGA solution per well to be stained.</li>
		<li>Incubate it in darkness at 4 &#176;C for 2 hr.</li>
		<li>Remove the unbound stain by washing the wells with 200 &#181;l of PBS three times.</li>
		<li>Let the plate dry for 15 min at RT.</li>
		<li>Dissolve the bound stain in 33% acetic acid, using 200 &#181;l per well.</li>
		<li>Seal the wells with strip caps and sonicate them using a water bath sonicator for 30 sec at RT and 40 kHz.</li>
		<li>Incubate the plate for 1 hr at RT.</li>
		<li>Repeat the sonication step. The wells can remain sealed between the sonication steps.</li>
		<li>Measure the fluorescence at &#955;ex = 495 nm and &#955;em = 520 nm with a plate reader (top reading).</li>
	</ol>
	</li>
	<li>Visualizing the matrix with fluorescence microscopy
	<ol>
		<li>Use the same protocol as above until step 6.1.6.</li>
		<li>After the drying step, visualize the samples with a fluorescence microscope, using a FITC filter (or another suitable green excitation filter).</li>
	</ol>
	</li>
</ol>
7. Staining Viable and Dead Cells within the Biofilms for Imaging with Fluorescence Microscopy and Quantification of the Signal for the Green-to-red-ratio (G/R)
<ol>
	<li>Imaging with fluorescence microscopy
	<ol>
		<li>Prepare a solution of each probe (one staining viable cells and the other one staining dead cells), according to the manufacturer&#39;s guidelines.</li>
		<li>Remove the planktonic solution from the wells and wash once with sterile PBS (200 &#181;l per well) using a multichannel pipette.</li>
		<li>Add 6 &#181;l of the staining solution per well.</li>
		<li>Incubate the plate in darkness for 15 min.</li>
		<li>Before the microscopy, remove the excess liquid manually using a multichannel pipette.</li>
		<li>Capture the images using a fluorescence microscope, using for instance a FITC filter (for green fluorescence, viable cells) or a TRITC filter (red fluorescence, dead cells).</li>
	</ol>
	</li>
	<li>Quantification of the signal as a green-to-red-fluorescence ratio (G/R)
	<ol>
		<li>Follow the same protocol as in steps 7.1.1 and 7.1.2.</li>
		<li>Add 200 &#181;l of the staining solution per well and incubate them for 15 min in darkness.</li>
		<li>Measure the fluorescence with a plate reader at excitation/emission wavelengths 485/535 nm and 485/635 for green and red fluorescence, respectively (top reading).</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

There is no single method that can simultaneously measure the effect of a compound on the viability, biomass, and biofilm matrix. Therefore, there is a need for combining assays in order to detect an effect on the three endpoints, preferably at a primary screening stage.
Resazurin is a very simple staining protocol consisting only of the addition of the redox probe. However, establishing the optimal incubation time of the biofilms with the resazurin is crucial to the success of this assay. In ...

Bacteria can switch between two very different lifestyles, planktonic and sessile, of which a biofilm is the most common example. In biofilms, bacteria form structured communities embedded in a self-produced matrix1. This self-produced matrix is a barrier between the bacteria and their external environment, and it protects the microbial cells, keeping them in close proximity. The composition of the biofilm matrix varies between and even within species, but it mostly consists of a tight network of lipopolysaccharides, extracellular DNA, and proteins. The matrix serves as a physical barrier inhibiting the entrance of harmful agents, but it also protects the biofilm from dehydration and prevents nutrients from escaping the cell2.
Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are purportedly responsible for over 80% of antibiotic-tolerant infections3. They display an inherently high tolerance against external threats: humidity, osmotic pressure, mechanical stress4, heat, UV radiation5, disinfectants, antimicrobial agents, and the host immune system1. For example, the required antimicrobial agent concentration required to kill a biofilm has been shown to be up to 1,000 times higher in comparison to that required to kill planktonic bacteria. The explanation for this higher tolerance seems to be multifactorial. The matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse; they transition between different metabolic states due to an existing gradient of oxygen, nutrients, and metabolites between the inner and outer parts of the biofilm6. Hence, in some biofilm regions, such as the core, bacteria are deprived of oxygen and nutrients and live in a metabolically less active or a completely dormant state7. The completely dormant cells are referred to as persister cells, and they are not susceptible to conventional antimicrobial treatments8. Thus, it is likely that biofilm resilience arises from a combination of the presently suggested and other, yet unknown, mechanisms. Staphylococcus spp. are still among the most problematic gram-positive bacteria, causing severe, often biofilm-related, infections4. It is suggested that up to 99% of all bacteria are associated within biofilms, making it the predominant bacterial lifestyle3. However, all of the currently existing antibiotics have been developed against single-cell (planktonic) bacteria. So far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place.
From a methodological perspective, additional challenges exist, as only a limited number of biofilm methods have been developed by standard-setting organizations, especially those applicable to the high-throughput screening of chemical libraries. All standardized assays (with only one exception) are based upon biofilm reactors, and these methods require large working volumes and large amounts of compounds to be tested, which are usually unavailable during the early investigational stage9-12. The only existing standardized screening-applicable assay is the so-called Calgary Biofilm Device, from which the commercially available minimum biofilm eliminating concentration (MBEC) system was developed13-15. However, the limitation of this assay is that the biofilms are grown on pegs, and not all bacterial species or even strains within the same species are able to form biofilms on this device. Moreover, methods that can be particularly applied to the exploration of natural compounds are needed. Natural products have been the major source for innovation in antimicrobial drug discovery over the past century16. They can provide novel anti-biofilm compounds with unique mechanisms of action that can also be effective against persister cells. Thus, the exploration of natural and naturally inspired libraries has high chances of producing promising and unique anti-biofilm leads.
Here, we present the experimental details of a platform of assays that was developed for the chemical screening of anti-biofilm compounds using three assays to measure the effects on the viability, total biomass, and matrix of Staphylococcus aureus biofilms. The first assay measures biofilm viability, and it is based on resazurin staining. Resazurin is a redox stain that is blue and non-fluorescent in its oxidized state and turns into pink, highly fluorescent resorufin when reduced by the metabolic activity of the bacteria. It is a very simple and fast method suitable for primary screenings17-20. The second assay, based on crystal violet staining, measures total biofilm mass. Crystal violet is a widely used stain for studying bacteria and bacteria in biofilms19,21-23. The assay is based on inexpensive reagents and has a simple absorbance endpoint reading. Finally, the third assay targets the extracellular polymeric substance (EPS)-matrix of the biofilm via wheat germ agglutinin (WGA), which binds specifically to poly-N-acetyl-glucosamine residues (PNAG) present in the matrix of staphylococcal biofilms24. The WGA is conjugated with a fluorophore that can be detected using fluorescence intensity readers25. We present here the rationale and details of the platform we developed, including examples of applications.

<table><tbody><tr><td>Resazurin</td><td>Sigma Aldrich</td><td>R7017</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma Aldrich</td><td>HT90132&amp;nbsp;</td><td></td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td><td>Thermo Fisher Scientific</td><td>W11261</td><td></td></tr><tr><td>LIVE/DEAD &lt;em&gt;BacLight&lt;/em&gt;&amp;nbsp;</td><td>Molecular Probes</td><td>L7012</td><td>SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red</td></tr><tr><td>Phosphate Buffered Saline</td><td></td><td></td><td></td></tr><tr><td>Tryptone soy agar</td><td>Lab M, Neogen</td><td>LAB011</td><td></td></tr><tr><td>Tryptine soy broth</td><td>Lab M, Neogen</td><td>LAB004</td><td></td></tr><tr><td>F96 Well Plate Polystyrene Sterile Clear Flat bottom</td><td>Thermo Fisher Scientific</td><td>161093</td><td></td></tr><tr><td>BRAND caps, strips of 8</td><td>Sigma Aldrich</td><td>BR781413-300EA</td><td></td></tr><tr><td>Branson CPX series ultrasonic bath</td><td>Sigma Aldrich</td><td>Z769428-1EA</td><td></td></tr><tr><td>Multipipette</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Multidrop dispenser</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Biomek 3000</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Varioskan Flash Multiplate reader</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC&amp;nbsp;</td><td>25923</td><td></td></tr></tbody></table>

a platform of anti-biofilm assays suited to the exploration of natural compound libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

In the proposed platform, the effects on the viability, biomass, and biofilm matrix are quantified. In the working sequence (Figure 1), one sample plate is stained with resazurin and subsequently with crystal violet to simultaneously evaluate the effects on bacterial biofilm viability and on total biofilm biomass. Both assays can be performed consecutively in the same plate because it was demonstrated earlier that a first staining with resazurin had no statistically signi...

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A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

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Research

JoVE Journal

Immunology and Infection

17.6K Views.  University of Helsinki. Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects. 

Video: A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

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 ...

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

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

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

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

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 remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

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

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

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Biology

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

A Solid Phase Adherence Assay to Assess Biofilm Formation

Source: Nickerson, K. P. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57322">Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification.</a>&#160;J. Vis. Exp.&#160;(2018)
The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

1. Preparation of Reagents

	Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Take a fresh 96-well plate, and add 100 microliters of CRPMI equilibrated to room temperature to each well of rows B to H. Dilute up to four test compounds separately in 1.0 milliliter of CRPMI to 2x the desired final concentration. Add 200 microliters of compound one in wells A1 to A3, 200 microliters of compound two in wells A4 to A6, 200 microliters of compound three in wells A7 to A9, and 200 microliters of compound four in wells A10 to A12.
Serially dilute the test compounds using a multichannel pipette. Begin by transferring 100 microliters from row A to row B, and mix. In that manner, continue to transfer 100 microliters well to well. Mix after each transfer and stop in row F. After mixing, expel 100 microliters from row F into a waste container. Do not transfer any liquid to rows G and H. Finally, add 100 microliters of CRPMI to each well of the plate using a multichannel pipette, to bring the final volume to 200 microliters.
Add 200 microliters of phosphate-buffered saline to a fresh sterile 96-well plate that features wells with dimensions identical to those of the biofilm initiation plate. Next, remove the plastic bag and paraffin film from the incubated biofilm initiation plate. Lift the lid straight up, avoiding contact between the pegs and the plate bottom as this may damage the biofilm. Keep the bottom part for subsequent OD600 analysis.
Rinse off planktonic cells by placing the peg lid onto the plate containing PBS. Submerge pegs for 5 seconds, lift them out of PBS, and submerge once more, being careful not to hit the pegs against the well sides. Place the rinsed peg lid into the challenge plate. Avoid unnecessary contact between pegs and the bottom plate, as this will damage the biofilm leading to inconsistent results.
Seal the plate with paraffin film, and place into a sealable plastic bag as before. Incubate the plate without shaking for 24 hours at 37 degrees Celsius in a 5% carbon dioxide incubator. Take the bottom of the biofilm initiation plate and resuspend the bacteria in each well using a multichannel pipette. Measure the OD600 with a suitable microplate reader to confirm growth occurring in all wells, but the sterility controls in row H.
Use a plate reader equipped with an incubation chamber, and capable of taking kinetic fluorescence readings for detecting resazurin conversion. Set up a 24-hour kinetic fluorescence measurement using a 530-nanometer excitation and 590-nanometer emission. Set the chamber temperature to 37 degrees Celsius and have the plate read every 20 minutes.
Set the plate reader to measure from the bottom of the plate, according to the manufacturer&#39;s instructions. Prepare to wash the plate by adding 200 microliters of PBS to each well of a sterile 96-well plate with a multichannel pipette. Then, prepare biofilm recovery medium by adding 400 microliters of 0.8 milligram per milliliter resazurin stock solution to 20 milliliters of CRPMI medium, to a final concentration of 16 micrograms per milliliter resazurin.
Next, add 150 microliters of biofilm recovery medium with a multichannel pipette, to each well of a fresh 96-well plate. Transfer the challenge plate from the incubator into a biosafety cabinet, and carefully remove the plastic bag and sealing film. Remove the peg lid from the challenge plate and rinse off planktonic cells in PBS as before, keeping the bottom of the challenge plate for subsequent OD600 analysis.
After rinsing, place the peg lid into the plate bottom containing the biofilm recovery medium. Wrap the side of the plate with paraffin film, being careful not to obstruct the bottom of the perimeter wells. Immediately after wrapping the plate, place it on the plate reader, and start a kinetic read over a 24-hour period by initiating the previously made resazurin kinetic protocol.

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Summary

Explore More Videos

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

Microtiter Dish Biofilm Formation Assay

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

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

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

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

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

A Solid Phase Adherence Assay to Assess Biofilm Formation

A Peg Plate Biofilm Assay for Screening Antibacterial Agents