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.
1. Biofilm Initiation
2. Preparation of the Biofilm Challenge Plate
3. Biofilm Challenge
4. Biofilm Determination
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.
The authors have nothing to disclose.
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 |