The Dilution Tests of Antimicrobials


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In a dilution test, the lowest dilution that inhibits turbidity (cloudiness) is the MIC. In this example, the MIC is 8 μg/mL. Broth from samples without turbidity can be inoculated onto plates lacking the antimicrobial drug. The lowest dilution that kills ≥99.9% of the starting inoculum is observed on the plates is the MBC. (credit: modification of work by Suzanne Wakim)

OpenStax Microbiology

The limitations of the Kirby-Bauer disk diffusion test do not allow for a direct comparison of antibacterial potencies to guide selection of the best therapeutic choice. However, antibacterial dilution tests can be used to determine a particular drug’s minimal inhibitory concentration (MIC), the lowest concentration of drug that inhibits visible bacterial growth, and minimal bactericidal concentration (MBC), the lowest drug concentration that kills ≥99.9% of the starting inoculum. Determining these concentrations helps identify the correct drug for a particular pathogen. For the macrobroth dilution assay, a dilution series of the drug in broth is made in test tubes and the same number of cells of a test bacterial strain is added to each tube. The MIC is determined by examining the tubes to find the lowest drug concentration that inhibits visible growth; this is observed as turbidity (cloudiness) in the broth. Tubes with no visible growth are then inoculated onto agar media without antibiotic to determine the MBC. Generally, serum levels of an antibacterial should be at least three to five times above the MIC for treatment of an infection.

The MIC assay can also be performed using 96-well microdilution trays, which allow for the use of small volumes and automated dispensing devices, as well as the testing of multiple antimicrobials and/or microorganisms in one tray. MICs are interpreted as the lowest concentration that inhibits visible growth, the same as for the macrobroth dilution in test tubes. Growth may also be interpreted visually or by using a spectrophotometer or similar device to detect turbidity or a color change if an appropriate biochemical substrate that changes color in the presence of bacterial growth is also included in each well.

The Etest is an alternative method used to determine MIC, and is a combination of the Kirby-Bauer disk diffusion test and dilution methods. Similar to the Kirby-Bauer assay, a confluent lawn of a bacterial isolate is inoculated onto the surface of an agar plate. Rather than using circular disks impregnated with one concentration of drug, however, commercially available plastic strips that contain a gradient of an antibacterial are placed on the surface of the inoculated agar plate. As the bacterial inoculum grows, antibiotic diffuses from the plastic strips into the agar and interacts with the bacterial cells. Because the rate of drug diffusion is directly related to concentration, an elliptical zone of inhibition is observed with the Etest drug gradient, rather than a circular zone of inhibition observed with the Kirby-Bauer assay. To interpret the results, the intersection of the elliptical zone with the gradient on the drug-containing strip indicates the MIC. Because multiple strips containing different antimicrobials can be placed on the same plate, the MIC of multiple antimicrobials can be determined concurrently and directly compared. However, unlike the macrobroth and microbroth dilution methods, the MBC cannot be determined with the Etest.

A microdilution tray can also be used to determine MICs of multiple antimicrobial drugs in a single assay. In this example, the drug concentrations increase from left to right and the rows with clindamycin, penicillin, and erythromycin have been indicated to the left of the plate. For penicillin and erythromycin, the lowest concentrations that inhibited visible growth are indicated by red circles and were 0.06 μg/mL for penicillin and 8 μg/mL for erythromycin. For clindamycin, visible bacterial growth was observed at every concentration up to 32 μg/mL and the MIC is interpreted as >32 μg/mL. (credit: modification of work by Centers for Disease Control and Prevention)
The Etest can be used to determine the MIC of an antibiotic. In this Etest, vancomycin is shown to have a MIC of 1.5 μg/mL against Staphylococcus aureus.

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology