A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Published: February 29, 2024

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 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. Incubate for 16 hr at 37 °C on a rotary shaker (100-200 rpm). Determine OD600 of the culture using a spectrophotometer and a st…

Representative 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 preparation of serial dilutions using a multichannel pipet. Concentrations are in µM.

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

Play Video

Bu Makaleden Alıntı Yapın
A Peg Plate Biofilm Assay for Screening Antibacterial Agents. J. Vis. Exp. (Pending Publication), e21980, doi: (2024).

View Video