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Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC)of antimicrobial substances
Irith Wiegand, Kai Hilpert & Robert E W Hancock
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Centre for Microbial Diseases and Immunity Research, University of British Columbia, 2259 Lower Mall Research Station, Vancouver, British Columbia, V6T 1Z4, Canada. Correspondence should be addressed to R.E.W.H. ([email protected]).
Published online 17 January 2008; doi:10.1038/nprot.2007.521
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 dened 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 dened period of time (1620 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.
/ INTRODUCTIONAgar dilution and broth dilution are the most commonly used techniques to determine the minimal inhibitory concentration (MIC) of antimicrobial agents, including antibiotics and other substances that kill (bactericidal activity) or inhibit the growth (bacteriostatic activity) of bacteria. The methods described here are targeted for testing susceptibility to antibiotic agents as opposed to other antimicrobial biocides such as preservatives and disinfectants.However, there are no major reasons why they cannot be used for these other antimicrobials. For agar dilution, solutions with dened numbers of bacterial cells are spotted directly onto the nutrient agar plates that have incorporated different antibiotic concentrations.After incubation, the presence of bacterial colonies on the plates indicates growth of the organism. Broth dilution uses liquid growth medium containing geometrically increasing concentrations (typically a twofold dilution series) of the antimicrobial agent, which is inoculated with a...