Name: in vitro biofilm formation in an 8-well chamber slide
Uploaded: 2011-01-20T18:30:00
Duration: 6 min 14 s
Description: Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

The work was funded by NIDCD/ NIH R01DC003915 to L.O. Bakaletz.

1. In Vitro Biofilm Formation
<ol>
<li>Bacterial colonies (which have been grown on appropriate agar overnight), were suspended in media and the OD490 was adjusted to 0.65</li>
<li>The resulting bacterial suspension was then diluted 1:6 (1 mL bacterial suspension + 5 mL pre-warmed medium) and incubated at 37&deg;C with 5% CO2 for approximately 3 hours in order to reach mid-log phase.</li>
<li>Dilute the mid-log growth suspension 1:2500 with pre-warmed media and place 200 &mu;L of dilution into each well of an 8-well chamber slide.</li>
<li>Incubate at 37&deg;C with 5% CO2.</li>
<li>After approximately 16 hours, aspirate medium from corner of each chamber and add 200 &mu;L fresh, pre-warmed medium - dispense along wall of chamber so as not to create shear forces within the chamber which could disrupt the biofilm.</li>
<li>If incubating longer than 24 hrs, change medium every 12 hrs or as needed to maintain bacterial viability.</li>
</ol>
2. 	Visualization of Biofilm
<ol>
<li>Aspirate medium from each chamber, as described above, and wash resident biofilm twice gently with sterile saline.</li>
<li>Add 200 &mu;L of BacLight Live/Dead stain (3 &mu;L component A + 3 &mu;L component B per mL of saline) to each well and incubate at room temperature for 15 minutes. Protect culture from light from this point onward.</li>
<li>Aspirate the stain and wash twice gently with sterile saline as before.</li>
<li>Add 200 &mu;L of neutral buffered formalin to each well and incubate for 30 minutes at room temperature to fix specimen.</li>
<li>Remove fixative and properly dispose of in accordance with institutional guidelines.</li>
<li>Wash twice with saline and dispose of the wash fluids that contain formalin as above.</li>
<li>Remove the plastic wells from the slide, add enough saline to each well so that when a coverslip is placed on the gasket, no air bubbles are present.</li>
<li>Coverslip and seal the edges of the coverslip with mounting medium. Nail polish can be used to seal the slides however the slides will not be as permanent.</li>
<li>Allow the sealant to dry for approximately 1 hour prior to examining via microscopy.</li>
</ol>
3. Results

 <img src="/files/ftp_upload/2481/2481fig1.jpg" alt="Figure 1" /> 
Figure 1. Representative 3D composite images of a biofilm formed in an 8-well chamber slide. Bacteria were labeled with Live / Dead stain wherein live bacteria fluoresce green and dead bacteria fluoresce red. (A) Composite image of entire biofilm showing distinct architecture with towers and water channels. (B) Same biofilm as in (A), now seen in cross section.

<ol>
	<li>Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+otitis+media:+evidence+and+relevance.">Bacterial biofilms in otitis media: evidence and relevance.</a> Pediatr Infect Dis J. 26, S17-S19 (2007).</li><li>Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li></ol>

No conflicts of interest declared.

Understanding the mechanism by which biofilms form, as well as characterizing their matrix components, is an area of intense interest. This protocol can be used to help identify proteins or other constituents that contribute to the structural integrity of the biofilm, as well as help identify and target proteins which may lend themselves to a therapeutic application.
The incubation times and dilutions in the above described protocol have been established for optimal biofilm formation by nontypeable Haemophilus influenzae (NTHI). Some preliminary work may be needed to optimize these steps for other bacterial species (i.e. initial dilution and incubation time needed to achieve mid-log phase growth). The 1:2500 dilution for the initial seeding of the chambers was established to ensure that a robust biofilm would develop in the chamber slides as induced by this organism and within the incubation times stated, without the biofilm overgrowing the chamber and/or exhausting available nutrients. Accordingly, the initial dilution and or incubation times may need to be adjusted for other bacterial species.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Chocolate II agar</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>B21267X</td>
<td>&nbsp;</td>
<tr>
<td>8-well chamber slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-656-18</td>
<td>&nbsp;</td>
<tr>
<td>BacLight Live/Dead bacterial viability kit</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9439023</td>
<td>&nbsp;</td>
<tr>
<td>BHI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>211059</td>
<td>&nbsp;</td>
<tr>
<td>Hemin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H5533</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-NAD</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N7004</td>
<td>&nbsp;</td>
<tr>
<td>Permaslip mounting media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9693613</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

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

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. ...

Research

JoVE Journal

Immunology and Infection

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 ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Kvantitative<em&gt; In vitro</em&gt; Assay til måling neutrofiladhæsion til Aktiverede primære humane mikrovaskulære endotelceller under statiske forhold

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Enteriske Bakteriel invasion af tarmepitelceller<em&gt; In Vitro</em&gt; Er dramatisk forøget hjælp af en lodret diffusionskammer Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Måling af ekstracellulær DNA udledning og kvantitativ NET Formation i humane neutrofiler<em&gt; In vitro</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Etablering af en High-throughput Opsætning til screening små molekyler, der modulerer c-di-GMP Signalering i<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Visualisere virkningerne af sputum på Biofilm Development Ved hjælp af en Chambered dækglas Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In vitro- metoder til sammenligning af målbinding og CDC-induktion mellem terapeutiske antistoffer: applikationer i biosimilaritetsanalyse

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

en<em&gt; In Vitro</em&gt; Caseum Binding Assay, der forudser lægemiddelpenetration i tuberkulose-læsioner

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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

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

Visualisering af Biofilmdannelse i Candida albicans ved hjælp af en automatiseret mikrofluid enhed

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Galde Salt-induceret Biofilmdannelse i enterisk patogener: teknikker til identifikation og kvantificering

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...