The minimum inhibitory concentration (MIC) of an antibacterial is defined as the maximum dilution of the product that will still inhibit the growth of a test microorganism. The minimum lethal concentration (MLC) of an antibacterial is defined as the maximum dilution of the product that will kill a test organism. MIC/MLC values can be determined by a number of standard test procedures. The most commonly employed methods are the tube dilution method and agar dilution methods. Serial dilutions are made of the products in bacterial growth media. The test organisms are then added to the dilutions of the products, incubated, and scored for growth. This procedure is a standard assay for antimicrobials. The procedure incorporates the content and intent of the American Society for Microbiology (ASM) recommended methodology.
Study Outline
The MIC portion of this test procedure begins with diluting the test product serially with equal volumes of 2X growth media. The resulting suspensions are 1X growth media with decreasing concentrations of the test product. The dilutions increase in such a way that the next dilution is half as concentrated as the dilution before (i.e. 1:2, 1:4, 1:8, 1:16:, 1:32...). Each dilution is then spiked with a of test culture and the dilutions are incubated.
It is important to keep in mind that MLC cannot be determined without an initial MIC test. After incubation, the MIC dilution tubes will either be turbid with growth or they will be clear. A tube without turbid growth will result from one of two possible events. Either the challenge microorganisms were killed by the test product, or they were inhibited by the test product. Negative tubes that are suspected of having inhibited microorganisms are subcultured in neutralizer broth. The neutralizer broth tubes are then incubated and scored for growth. A subculture tube that becomes turbid after incubation indicates that the test product was inhibitory at that dilution for that particular organism. If the subculture tubes remain negative after incubation, this indicates that the test product is lethal at that concentration for that particular organism.
Test products that are not clear or precipitate the growth media follow the same basic test procedure described above, but in order to detect growth of the test organisms, each dilution must be plated onto agar and incubated.
Sample Requirements
The tube dilution procedures are limited to products which do not precipitate or cloud the growth media within the expected endpoint range.
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- Kimberlee Dickman 801-290-7858, 800-826-2088
