This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).

1. HSV Strains and Handling
Note: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ- with beta-galactosidase reporter activity used were provided by V. Twiari36,37.
<ol>
	<li>Use virus from a single lot and store at -80 &#176;C at a 1:1 ratio of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 20% fetal bovine serum (FBS) and skim milk until use. Before viral lot storage, determine virus concentration by o-nitrophenyl-&#946;-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal) assay.</li>
	<li>Determine virus viability and multiplicity of infection (MOI) by X-Gal staining for each experimental assay run using reporter virus entry assay, as previously described (Figure 1)14.</li>
	<li>Dilute virus (Opt-MEM) to desired MOI. Fix monolayers (paraformaldehyde; 0.5 ml/well) before staining. Place virus viability controls in a separate microtiter plate in parallel with polymicrobial assay plates.</li>
</ol>
2. HeLa 299 Cell Handling
<ol>
	<li>Grow at 37 &#176;C, 5% CO2 in DMEM with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (FBS), gentamicin (50 &#181;g/ml) and L-glutamine. Passage cells at 80% confluence with Trypsin solution (ethylenediaminetetraacetic acid, 0.53 M EDTA; 0.05% Trypsin; 5 ml/flask).</li>
</ol>
3. C. albicans Handling
Note: C. albicans obtained from a clinical laboratory source is stored at -80 &#176;C in Remmel skim milk 2x medium.
<ol>
	<li>Culture frozen stock onto Sabouraud Dextrose Medium (37 &#176;C). After 24 hr subculture the C. albicans onto Fungisel medium (37 &#176;C; 48 hr) for use.</li>
	<li>Generate germ tube (GT) forms (pick representative colonies; 3 ml FBS; 3 hr; 37 &#176;C; Abs600, 0.3). After incubation, wash GT (HBSS; 2x; 4,000 x g). Add washed GT to warmed HBSS (37 &#176;C; 0.32 Abs600). GT forms should be 99% of cells observed as determined by hemocytometer count.</li>
	<li>Make yeast form (YF) stock suspensions by picking representative Fungisel colonies (HBSS, 3 ml; 0.32 Abs600). Count the number of YF forms/ml microscopically using a hemocytometer. YF forms should be 99% of cells observed as determined by hemocytometer count.</li>
	<li>Make working fungal stock (250 &#181;l of GT or YF stock in 25 ml HBSS; 37 &#176;C; 105 CFU/ml)</li>
</ol>
4. S. aureus Handling
<ol>
	<li>Store S. aureus ATCC 25923 (-80 &#176;C; Remmel skim milk 2x). Culture onto sheep blood agar (5%; 37 &#176;C; 24 hr). Pick representative colonies and transfer to mannitol salts medium within 2 days for stock (37 &#176;C; 18 hr).</li>
	<li>Make S. aureus stock suspension (3 ml HBSS; 1.32 Abs600 ;108 CFU/ml)
	<ol>
		<li>Make working S. aureus stock (100 &#181;l of the stock in 25 ml HBSS; 105 CFU/ml).</li>
	</ol>
	</li>
</ol>
5. Candida and S. aureus Suspensions
<ol>
	<li>Make mixed C. albicans and S. aureus suspension (250 &#181;l YF or GT stock and 100 &#181;l S. aureus stock in 25 ml HBSS).</li>
</ol>
6. Polymicrobial Biofilm Assay
<ol>
	<li>Seed 96 well plates with 200 &#181;l of 2 x 105 HeLa cells/ml (85% confluence level). Rock plates (30 - 45 min; 37 &#176;C) before incubation (37 &#176;C; 5% CO2 incubator; 18 hr). Wash monolayers (1x; Opt-MEM ) then seed with HSV (HSV-1 (KOS) gL86 or HSV-2 (KOS) 33 gJ- in 100 &#181;l Opt-MEM ; MOI 50 and 10). Incubate plates (3 hr; 37 &#176;C; 5% CO2). Use only one viral strain per day.</li>
	<li>Wash infected monolayers (1x; phosphate buffered saline (PBS) with Mg+2 and Ca+2; 100 &#181;l). Replace PBS with warm HBSS leaving 25 &#181;l in each well.
	<ol>
		<li>Add YF, GT and/or S. aureus working suspensions (100 &#181;l; target to cell ratio =5:1; n=16) as indicated in Table 1. Incubate plates (static; 30 min; 37 &#176;C; 5% CO2).</li>
	</ol>
	</li>
	<li>After incubation, aspirate one column at a time immediately refilling with 300 &#181;l PBS with Mg+2 and Ca+2. Repeat this step twice then add radio-immunoprecipitation assay lysis buffer (RIPA; filter sterilized; 200 &#181;l of a 1:50 dilution).</li>
	<li>Rapidly triturate the HeLa cell lysate then place 50 &#181;l onto mannitol salts (MS) and/or Fungisel (F) media (Figure 2). Spread the lysate using a glass rod bent at a 90&#176; angle. Incubate the plates (18 hr at 37 &#176;C). Manually count the number of colonies per plate. Controls consist of S. aureus and/or C. albicans adherence to HSV-uninfected HeLa cells.</li>
</ol>
7. Imaging Studies
<ol>
	<li>Wash each round glass coverslip (12 mm; 50 ml acetone in 100 ml beaker). Dry and sterilize coverslips (Kimwipes; glass petri dishes). Place dry sterile coverslips into the wells of sterile 24 well plates with alcohol flame sterilized forceps.</li>
	<li>Add HeLa cells (1 ml; 5x volume used for 96 well plates) to the wells of 24 well plates containing the washed sterile round glass coverslips. Add the virus, bacteria and fungi according to the template (Table 2) at 5x the volume used for the 96 well plates, then incubate and process as described in steps 6.2.1 to 6.4 above.</li>
	<li>After the final wash, fix the cells for microscopy by flooding the slide with methanol and allowing it to evaporate. Store the plates at RT until staining.</li>
	<li>For bright field microscopy (1,000x final original magnification) fill the wells containing methanol-fixed coverslips with deionized water. Immediately aspirate water. Cover each coverslip with Grams crystal violet. Wash coverslips free of non-bound stain (deionized water). Dry coverslips in situ then adhere them with hard set mounting medium to a labeled slide (Figure 3).</li>
	<li>For fluorescent microscopy (100x objective; 1,000x final original magnification) wash coverslips free of methanol essentially as described in step 7.4. Dry the coverslips in the wells.
	<ol>
		<li>After drying, remove the cover slips and place them on labeled slides. Then add a sufficient amount of 1:20 dilution of fluorescein isothiocyanate (FITC)-conjugated Herpes Simplex Virus Type 1 + 2 gD antibody to cover the coverslip (1 - 5 &#181;l).</li>
		<li>Incubate the slides in a moisture chamber at 37 &#176;C for 30 min. After incubation, wash the coverslips in 4 changes of PBS.</li>
		<li>After the final wash, return the coverslip to the labeled slide. Stain with 4&#8242;,6-diamidino-2-phenylindole (DAPI; moisture chamber; 37 &#176;C for 30 min). After incubation wash the coverslips in 4 changes of PBS, then affix to the labeled slide with hard set mounting medium.</li>
		<li>Allow the mounting medium to cure for 24 hr at RT in the dark. Examine the coverslips under oil objective on either a bright field microscope or fluorescent microscope with FITC and DAPI cutoff filters. Examine pictures of at least 50 fields (100 cells per organism minimum) for co-localization (Figure 4).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Currently no information is available on complex interactions between permanent to semi-permanent members of the host microbiome that cross multiple taxonomic domains, i.e., prokaryotic, eukaryotic and viral. Therefore we developed a novel in vitro model system to study biofilm initiation by S. aureus and C. albicans on HSV-1 or HSV-2 infected HeLa 229 (HeLa) cells 38. The HeLa cell model system presents a unique advantage. This is due to their lack of surface fibronectin ex...

The human microbiome includes diverse organisms from multiple taxonomic kingdoms that share geographic regions in the body. Adherence to cell surfaces is an essential first step in biofilm formation, which is part of the microbiome colonization process. Included in the microbiome can be viruses that cause chronic and persistent infections. The chronic cell infection by these viruses can cause an alteration in putative receptor availability.1,2 In addition, cell entry by intracellular pathogens could also affect host membrane fluidity/hydrophobicity which in turn may alter attachment of other microbiome members, including bacteria and fungi. In order to understand the interactions that can occur between these multiple pathogens that co-localize in the same geographic regions of the human host, we must be able to study the interaction of pathogens that represent the spectrum of taxonomic kingdoms present at the mucosal surface.
The Herpesviridae are a family of microbes present in 100% of humans as permanent members of the microbiome3,4. In addition they can also be persistently shed both in the presence and absence of symptoms. Specifically, herpes simplex virus-1 and herpes simplex virus-2 (HSV-1 and HSV-2, respectively) are permanent members of the microbiome in the oronasopharynx and genital tract. In immune-competent individuals, both HSV-1 and HSV-2 cause gingivostomatitis, as well as genital herpes5-8. At these sites, HSV causes a latent infection characterized by chronic persistent asymptomatic viral shedding9. Entry of HSV into cells results in alterations in surface expression of nectins, heparan sulfate, lipid rafts and herpesvirus entry mediator/tumor necrosis factor receptor (HVEM/TNFr)10-25. These potentially represent shared receptors for some bacteria and fungi, e.g. S. aureus and C. albicans,which while opportunistic pathogens, can also reside as members of the mucosal microbiome of the oronasopharynx 26,27. Within the oronasopharynx S. aureus and C. albicans occupy two distinct sites of colonization. In hosts with natural teeth, the oral mucosa is shared by HSV-1 and C. albicans, while the anterior nasal nares are occupied by S. aureus28. However, despite in vitro findings that S. aureusadheres to mouth epithelial cells, 29,30 S. aureus is infrequently isolated from oral specimens when normal tissue is present29,30. Little is known concerning genital tract co-colonization niches beyond the clinical findings that S. aureus is associated with aerobic vaginitis, characterized by genital inflammation, discharge and dyspareunia, while C. albicans produces mucosal lesions similar to that observed in the oral cavity31-35. Thus, although these members of the oral and genital microbiome cross taxonomic kingdoms little is known concerning their interaction as it impacts their ability to initiate biofilm formation through adherence to the host cell surface5. This protocol has been effectively applied to determine the functional consequences of co-colonization/infection.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;C.albicans&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BBL Sabouraud Dextrose</td><td>BD</td><td>211584</td><td></td></tr><tr><td>Fungisel Agar</td><td>Dot Scientific</td><td>7205A</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;S.aureus&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mannitol Salt Agar</td><td>Troy Biologicals</td><td>7143B</td><td></td></tr><tr><td>Sheep blood agar</td><td>Troy Biologicals</td><td>221239</td><td></td></tr><tr><td>&lt;strong&gt;Hela cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x DMEM (Dubelcco&#039;s Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td><td>Corning</td><td>10-017-CM</td><td></td></tr><tr><td>Gentamicin 50 mg/ml</td><td>Sigma</td><td>1397</td><td>50 &amp;micro;g/ml final concentration in the complete DMEM</td></tr><tr><td>Trypsin EDTA (0.05% Trypsin, 0.53 M EDTA)Solution 1x</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% final concentration in the complete DMEM</td></tr><tr><td>&lt;strong&gt;Other medium and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ONPG</td><td>Thermo Scientific</td><td>34055</td><td></td></tr><tr><td>Ultra-Pure X gal</td><td>Invitrogen</td><td>15520-018</td><td></td></tr><tr><td>1x HBSS (Hanks&#039; Balanced Salt Solution)</td><td>Corning</td><td>20-021-CV</td><td></td></tr><tr><td>1x PBS</td><td>Dot Scientific</td><td>30042-500</td><td></td></tr><tr><td>RIPA Lysis</td><td>Life Technologies</td><td>89901</td><td></td></tr><tr><td>&lt;strong&gt;Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A433P-4</td><td></td></tr><tr><td>HSV 1&amp;amp;2, specific for gD</td><td>ViroStat</td><td>196</td><td></td></tr><tr><td>DAPI</td><td>SIGMA</td><td>D8417-5MG</td><td></td></tr><tr><td>Gram Crystal Violet</td><td>Troy Biologicals</td><td>212527</td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Petri dish 100 x 15</td><td>Dot Scientific</td><td>229693&amp;nbsp;</td><td></td></tr><tr><td>Petri dish 150 x 15</td><td>Kord Valmark</td><td>2902</td><td></td></tr><tr><td>96-Well plates</td><td>Evergreen Scientific</td><td>222-8030-01F</td><td></td></tr><tr><td>24-well plates</td><td>Evergreen Scientific</td><td>222-8044-01F</td><td></td></tr><tr><td>Culture tubes 100 x 13</td><td>Thomas Scientific</td><td>9187L61</td><td></td></tr><tr><td>Cover slip circles, 12 mm</td><td>Deckglaser</td><td>CB00120RA1</td><td></td></tr></tbody></table>

determination of biofilm initiation on virus-infected cells by bacteria and fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

The level of robustness of data obtainable from system described in this report is shown in Figure 2 a-f 38. Through the use of this system the modulation of staphylococcal and fungal interaction with virally infected cells and their effect on each other&#39;s adherence can be delineated. These types of studies require microscopic examination of the interaction as shown in Figures 3 and 4 38 in order to determine whe...

Watch this Scientific Journal Video about Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi at JoVE.com

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

determination-biofilm-initiation-on-virus-infected-cells-bacteria

Research

JoVE Journal

Immunology and Infection

9.6K Views.  Midwestern University. A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Video: Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Biology

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

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 ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

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. ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://app.jove.com/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

Source: Guennoc, Cora Miquel, et al, <a href="https://app.jove.com/v/54771">A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.</a> J. Vis. Exp. (2017).
This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony

	Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in ...

An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells

Source: Plotkin, B. J. et al., <a href="https://app.jove.com/v/54162">Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi.</a> J. Vis. Exp. (2016)
This video illustrates the initiation of polymicrobial biofilm formation on virus-infected cells by bacteria and fungi. Herpes simplex virus infection impedes bacterial attachment to the cells, preventing bacterial-fungal co-localization. In contrast, uninfected cells display bacterial-fungal co-localization and initiation of biofilm formation.

1. HSV Strains and Handling
NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ-&#160;with beta-galactosidase reporter activity is used ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Samenvatting

Meer video's verkennen