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Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances


The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16–20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.

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Figure 1
Figure 2
Figure 3: Outline of the setup of a microtiter plate for antimicrobial susceptibility testing with doubling dilutions of eight different antimicrobial agents in two different concentration ranges with labeling suggestions in blue.
Figure 4: Interpretation of possible growth patterns in MIC microtiter plates.


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We acknowledge the financial assistance of the Applied Food and Materials Network and the Canadian Institutes of Health Research. R.E.W.H. was supported by a Canada Research Chair award. K.H. was supported by a fellowship from the Canadian Institutes of Health Research. I.W. was supported by the Juergen-Manchot-Foundation.

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Correspondence to Robert E W Hancock.

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Wiegand, I., Hilpert, K. & Hancock, R. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3, 163–175 (2008).

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