A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Abstract

Source: Dalecki, A. G., et al. Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay. 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's metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

Protocol

1. Biofilm Initiation

1. Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate Staphylococcus aureus strain Newman in 10 ml of Mueller-Hinton medium from a glycerol stock. Perform all work involving handling of S. aureus with gloves and within a biosafety cabinet.
2. Incubate for 16 hr at 37 °C on a rotary shaker (100-200 rpm).
3. Determine OD₆₀₀ of the culture using a spectrophotometer and a standard cuvette (path length: 1cm).
4. Calculate the volume (V) of culture needed to prepare 20 ml of inoculum with OD_{600(inoculum)} of 0.1 in chelexed Roswell....

Results

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mueller-Hinton Medium	Oxoid	CM0405	Follow recommendations of manufacturer
RPMI-medium	Corning	17-105-CV
CRPMI	Ref 9		RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640
Chelex 100 Resin	Bio Rad	142-2822
MBEC-plates	Innovotech	19111
Resazurin Sodium Salt	Sigma	R7017	800 µg/ml in DI water Filter sterile
Micro Plate Shaking Platform	Heidolph Titramax 1000
Cytation 3 Plate Reader	Biotek
Gen5 software	Biotek		Recording and analysis of resazurin conversion
Neocuproine	Sigma	N1501	Prepare 10 mM stock in 100% Ethanol, store at -80 ºC
Copper sulfate	Acros Organics	7758-99-8	Prepare a 100 mM stock solution in water, store at 4 ºC
Cu-Neocuproine	Self-Made		Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.
Gentamicin	Sigma	G3632-1G