The minimum inhibitory concentration (MIC) is the smallest concentration of an antimicrobial agent that inhibits the growth of bacteria. The value is obtained in a highly mechanized fashion, but this procedure interval-censored reading. It is often of interest to use data collected from complex experiments to see how the mean MIC is affected by different factors.
Principles:
The Minimum Inhibitory Concentration Assay is a technique used to determine the lowest concentration of a particular antibiotic needed to kill bacteria. This assay is typically performed on planktonic (free-floating) bacterial cells.
Methods for determination of MIC:
- Serial/Broth Dilution method
- Agar Dilution Method
Material used:
- Laminar air flow
- Petri dishes
- Tips
- Micropipettes
- Muller Hinton Broth and Agar 1.5%
- Vortex Mixer
- Multi-inoculator
Procedure:
Serial/Broth Dilution
Broth dilution testing allows the option of providing both quantitative (MIC) and qualitative (category interpretation) results. MIC can be helpful in establishing the level of resistance of a particular bacterial strain and can substantially affect the decision to use certain antimicrobial agents.
Broth dilution can again be performed by 2 ways
- Macro dilution: Uses broth volume of 1 ml in standard test tubes .
- Microdilution: Uses about 0.05 to 0.1 ml total broth volume and can be performed in a microtiter plate or tray .
The procedure for both macro and microdilution are same except the volume of the broth.
For example, let’s say you wish to determine the MIC of an antibiotic on a bacterium. You decide to test 3 concentrations (10 µg/ml, 1 µg/ml and 0.1 µg/ml). Each of these tubes have growth media inoculated with a standard concentration of bacteria and the respective antibiotic concentration (Figure 1).
The tubes are allowed to incubate overnight. Broth tubes that appear turbid are indicative of bacterial growth while tubes that remain clear indicate no growth. The MIC of the antibiotic is the lowest concentration that does NOT show growth.
After incubating the tubes overnight, you observe the tubes in Figure 2:
From this example, Tube C, (with 0.1 ug/ml of antibiotic) did not inhibit bacterial growth. Tubes A and B, on the other hand, did inhibit growth. Tube B has the minimum inhibitory concentration because B is the lowest concentration of the antibiotic that inhibited cell growth. Therefore, the MIC for this bacterium is 1 µg/ml.
Agar dilution
MIC of an antibiotic using broth dilution method is determined by using the following procedure
- Preparation of antibiotic stock solution
- Preparation of antibiotic dilution range
- Preparation of agar dilution plates
- Preparation of inoculum
- Inoculation
- Incubation
- Reading and interpreting results
Antibiotic stock solution can be prepared by commercially available antimicrobial powders (with given potency). The amount needed and the diluents in which it can be dissolved can be calculated by using either of the following formulas to determine the amount of antimicrobial powder (1) or diluent (2) needed for a standard solution:
Prepare antimicrobial agent stock solutions at concentrations of at least 1000 μg/mL (example: 1280 μg/mL) or 10 times the highest concentration to be tested, whichever is greater.
Because microbial contamination is extremely rare, solutions that have been prepared aseptically but not filter-sterilized are generally acceptable. If desired, however, solutions may be sterilized by membrane filtration. Dispense small volumes of the sterile stock solutions into the sterile glass, polypropylene, polystyrene, or polyethylene vials; carefully seal; and store (preferably at −60 °C or below, but never at a temperature warmer than −20 °C and never in a self-defrosting freezer). Vials may be thawed as needed and used the same day.
Preparation of antibiotic dilution range
- Use sterile 13- x 100-mm test tubes to conduct the test. If the tubes are to be saved for later use, be sure they can be frozen.
- Close the tubes with loose screw-caps, plastic or metal closure caps, or cotton plugs.
- Prepare the final two-fold (or other) dilutions of antimicrobial agent volumetrically in the broth. A minimum final volume of 1 mL of each dilution is needed for the test.
Note: For, microdilution, only 0.1 ml is dispensed into each of the 96 wells of a standard tray.
Preparation of inoculum
- Prepare the inoculum by making a direct broth suspension of isolated colonies selected from an 18- to 24-hour agar plate (use a non-selective medium, such as blood agar).
- Adjust the suspension to achieve turbidity equivalent to a 0.5 McFarland turbidity standard. This results in a suspension containing approximately 1 to 2 x 10^8 colony forming units (CFU)/mL for Escherichia coli ATCC® 25922.
- Compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines.
- Optimally within 15 minutes of preparation, dilute the adjusted inoculum suspension in broth so, after inoculation, each tube contains approximately 5 x 10^5 CFU/mL. Note: This can be accomplished by diluting the 0.5 McFarland suspension 1:150, resulting in a tube containing approximately 1 x 10^6 CFU/mL. The subsequent 1:2 dilution in step 3 brings the final inoculum to 5 x 10^5 CFU/mL.
Inoculation
Within 15 minutes after the inoculum has been standardized as described above, add 1 ml of the adjusted inoculum to each tube containing 1 ml of the antimicrobial agent in the dilution series (and a positive control tube containing only broth), and mix.
This results in a 1:2 dilution of each antimicrobial concentration and a 1:2 dilution of the inoculums.
Incubation:
Incubate the inoculated tubes at 35 ± 2 ºC for 16 to 20 hours in an ambient air incubator. To maintain the same incubation temperature for all cultures, do not stack microdilution trays more than four high.
Interpretation
Compare the amount of growth in the wells or tubes containing the antimicrobial agent with the amount of growth in the growth-control wells or tubes (no antimicrobial agent) used in each set of tests when determining the growth endpoints. For a test to be considered valid, acceptable growth (≥ 2 mm button or definite turbidity) must occur in the growth-control well.
Quality Control
Use reference bacterial strains that are genetically stable and have well-defined MICs that are in the middle range of the expected MICs of each antimicrobial agent to be tested. A dilution series should include at least two concentration increments above and below the previously established MIC for the reference organisms.
CLSI has established QC limits for dilution susceptibility tests; an unacceptable QC result is one that falls outside these published limits. Reference strains recommended by the CLSI for QC of dilution tests for aerobic bacteria are
E. coli ATCC 25922,
E. faecalis ATCC 29212,
P. aeruginosa ATCC 27853, and
S. aureus ATCC 29213.
Troubleshooting Microdilution Assays
Inappropriate MICs report:
When MICs are lower than expected—the inoculum may be too light.
When MICs are higher than expected—the inoculum may be too heavy.
In such conditions, repeat testing using McFarland 0.5 turbidity standard or standardizing device. Check steps in inoculum preparation and inoculation procedure.
When MICs are either higher or lower than expected—the composition of the cation-adjusted Müeller–Hinton broth may not be optimal. Check the pH and calcium concentration of in-house prepared media. Use an alternative commercial lot of media or an alternative lot of commercial panels.
When there are skipped wells—may be caused by several problems:
Check for contamination.
A panel may have been inadequately inoculated or the inoculum may have been inadequately mixed.
Drug concentration in the wells may be inaccurate.
Volume of broth in the wells may be inaccurate.
When several MICs too high or too low—they may indicate a possible reading/transcription error. Recheck all of the readings and repeat testing using an alternative lot.
