eoe sub articles

EoE Sub Articles

1. Biofilm Initiation
<ol>
	<li>Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman in 10 ml of Mueller-Hinton medium from a glycerol stock. Perform all work involving handling of&#160;S. aureus&#160;with gloves and within a biosafety cabinet.</li>
	<li>Incubate for 16 hr at 37 &#176;C on a rotary shaker (100-200 rpm).</li>
	<li>Determine OD600&#160;of the culture using a spectrophotometer and a st...

a peg plate biofilm assay for screening antibacterial agents

Source: Dalecki, A. G., et al. <a href="https://app.jove.com/v/53925">Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay.</a> J. Vis. Exp. (2016)
This video demonstrates an assay for screening antimicrobial compounds that efficiently eliminate biofilms using a peg-plate. The methodology monitors the biofilm&#39;s metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

<img alt="Figure 1" src="/files/ftp_upload/21980/21980_Figure1.jpg" /> 
Figure 1. Challenge plate layout. Example layout of challenge plate accommodating parallel testing of four compounds in triplicate at six distinct concentrations. Untreated and sterility controls are also included. The layout design supports convenient in-plate preparation of serial dilutions using a multichannel pipet. Concentrations are in &#181;M.

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

1. Biofilm Initiation

	Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman ...

Take a microtiter plate with a lid featuring cylindrical supports, or pegs, coated in bacterial biofilms.
Place the biofilm-coated peg-lid onto microtiter plate wells with buffer to eliminate loosely attached or planktonic bacteria, focusing on biofilm-attached measurements.
Transfer the rinsed biofilm-coated peg-lid to a challenge plate with increasing test antibacterial agent concentrations. Incubate without shaking.
Depending on the agent&#39;s concentration and antibacterial potential, it disrupts the bacterial metabolic activity and biofilm integrity.
Post-incubation, place the peg lid onto buffer-containing plate wells to remove planktonic bacteria.
Transfer it to microtiter plate wells with a biofilm recovery medium containing resazurin, a cell-permeable, weakly fluorescent dye. Incubate.
In metabolically active bacteria, dehydrogenase enzymes reduce blue-colored resazurin into pink-colored, fluorescent resorufin.
Lack of resazurin conversion indicates biofilm eradication, while conversion to resorufin suggests the presence of viable cells.
The lowest test antibacterial agent concentration yielding no resazurin conversion denotes the minimal biofilm-eradicating concentration.

a-peg-plate-biofilm-assay-for-screening-antibacterial-agents

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

JoVE Journal

Environment

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 ...

Immunology and Infection

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

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