Name: visualizing the effects of sputum on biofilm development using a chambered coverglass model
Uploaded: 2016-12-14T14:00:00
Description: Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

TB acknowledges a research fellowship from Cystic Fibrosis Canada.

Note that Research Ethics Board (REB) is required to collect and store sputum samples from human subjects. These studies were approved by the Hospital for Sick Children REB#1000019444.
1. Preparing CF Sputum Samples
<ol>
	<li>Collect sputum sample from patients during routine visits to the cystic fibrosis clinic and keep on ice.</li>
	<li>Transport sputum sample on ice within the first hour of collection, to the research laboratory, to undergo processing.</li>
</ol>
2. ...

<ol>
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The methods described herein allow for visualization of bacterial biofilms grown in the presence of exogenous products. Not surprisingly, the production of the exoproducts is of importance when using this type of system. For instance, Dithiothreitol (DTT), is often used on human sputum samples to help liquefy the samples. However, the effect of DTT alone can decrease biofilm development and viability (data not shown). Thus, proper controls for all conditions are necessary. Furthermore, the addition of human sputum produc...

Bacterial biofilms are groups of microorganisms that are attached to one another and encased in a self-secreted matrix.1,2 Classically, they represent bacteria physically attached to an abiotic or biotic surface formed under conditions of flow. Biofilms have also been shown to grow in static conditions (absence of flow) and distal from surfaces, such as at the air-liquid interface of thermal pools or pellicles formed in test tubes. These biofilms have long been recognized in the environment and are a major detriment to industrial processes, as they can form in water reservoirs or in pipes, resulting in biofouling, corrosion and blockages.3,4
Biofilms are also critical in healthcare settings, as they have been shown be involved in catheter related infections, pulmonary infections in cystic fibrosis patients, as well as in numerous other infections.5,6 One of the hallmarks of biofilm infections is the decreased susceptibility of bacteria to antibiotics and impaired clearance by the innate immune system.7-9 The most well studied, clinically relevant scenarios involving biofilm-based infection occurs in patients with cystic fibrosis (CF), who are chronically infected with Pseudomonas aeruginosa biofilms. P. aeruginosa can undergo a number of changes during establishment of chronic infection that make it very difficult to treat.10,11 Biofilms can differentially activate innate immunity and drive inflammation.12-14 As these infections lead to increased morbidity and mortality in CF patients, it is crucial to understand factors that can affect biofilm development in this context.
A recent study suggests that host-factors are critical in the formation of P. aeruginosa biofilm aggregates.15 These biofilms contribute to reduced susceptibility to antibiotics and host defense mechanisms. The presence of host-derived factors, such as neutrophil elastase, as well as secreted products from microorganisms present in the CF lung, have the potential to greatly modulate biofilm formation and development.16 Additionally, biofilms interact with the host to modulate expression of numerous pathways and initiate inflammation. While high throughput methods, such as the standard crystal violet assay, can provide some information with regards to the biofilm process, visualization of the biofilm in response to these factors provide more in-depth information.
In this manuscript we describe a method for using factors from the sputum of patients with CF to study the development of biofilms in vitro. This method allows for rapid visualization of biofilms exposed to sputum containing host factors using a commercial biofilm viability kit. This technique can be used to visually identify changes that occur during biofilm growth in the presence of exogenous products, and represents an improved method to analyze the changes in biofilm development under various conditions.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td>
			<td style="width:159px;">Thermo Sicher Scientific</td>
			<td style="width:136px;">155409</td>
			<td style="width:729px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Filmtracer Live/Dead Biofilm Viabilty Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">L10316</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Blood agar plates</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">R10215</td>
			<td>Confirming viability via CFU counts or selecting colonies for innoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">COMSTAT</td>
			<td style="width:159px;">Availble software online</td>
			<td style="width:136px;"></td>
			<td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Millers LB Broth</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>12780-052</td>
			<td style="width:729px;">Standard media for overnight gowth/biofilm growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex-GV Syringe Filters</td>
			<td style="width:159px;">Millipore</td>
			<td>SLGV013SL</td>
			<td>Filtering of sputum supernants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Phosphate Buffered Saline (Dulbecco A)</td>
			<td style="width:159px;">Oxoid</td>
			<td style="width:136px;">BR0014G</td>
			<td>Washing of biofilm chambers after media removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss AxioVert 200M</td>
			<td style="width:159px;">Carl Zeiss</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamamatsu C9100-13 EM-CCD</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectral Borealis</td>
			<td style="width:159px;">Qs Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perkin Elmer Volocity</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td>
		</tr>
	</tbody>
</table>

visualizing the effects of sputum on biofilm development using a chambered coverglass 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 development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

The overall design of the experiment is represented in Figure 1. The use of this protocol provides a convenient method to visualize the changes in biofilms grown for different periods of time (e.g., 24, 48 or 72 hr). Importantly, exogenous signals, such as sputum filtrates, can be added to visualize the changes in biofilm development. As seen in Figure 2, the presence of 10% sputum filtrates can change the architecture of the biofilm (Fig...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

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

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

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 ...

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