DETERMINATION OF MIC

 

Determination of Minimal Inhibitory Concentration

AIM: To identify the MIC of given antimicrobial compound or antibiotic stock solution.

PRINCIPLE: The agar dilution technique is used to measure qualitatively the in vitro activity of an antimicrobial agent against the test bacteria. In this method, graded amounts of antibiotics are incorporated in agar plates and inoculated in spots with the organisms under study. If the organism under study is susceptible to the incorporated antibiotic, no bacterial growth is expected in agar plates with higher amounts of the drugs. Bacterial growth is observed as the antibiotic concentration in the agar plate diminishes. Inhibition of growth at the minimum or lowest concentration of antibiotic is regarded as the end point.

MATERIALS REQUIRED: Culture media, solvents, antimicrobial agents, control strains and apparatus needed for the minimal inhibitory concentration (MIC) test.

PROCEDURE:

1.      Dissolve the antimicrobial agent powder in solvent to make a concentration of 1,000 µg/ml.

2.      Dispense the stock solution into sterile diluents using two-fold dilution technique.

3.      Prepare MHA and Keep in a water bath at 48-50°C until use.

4.      Label each empty sterile plate in order to identify the antimicrobial agent and their concentrations.

5.      Pipette 1 ml of appropriate dilutions of the test antimicrobial agent into the labeled plate. Two replicates must be made for each concentration.

6.      Pipette 9 ml of MHA (keep warm at 48- 50°C), add into the plate with appropriate dilution of the test antimicrobial agent and mix thoroughly.

7.      Allow the agar to solidify at room temperature and Control agar plates/Drug-free agar plates

8.      Do not add any antimicrobial agent. There should at least be 2 control plates.

9.      From a pure 18-24 hour bacterial culture get 4- 5 isolated colonies.

10.  Shake vigorously in a water bath at 30°C until it achieves or exceeds the turbidity of 0.5 MacFarland standard (prepared by adding 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.36 NH2 SO4 ; commercially available). The inoculum may also be standardized based on optical density [OD625 of 0.08-0.1 (1cm light path)] using a spectrophotometer. This is usually achieved after 18- 24 hours.

11.  Dilute the standardized inoculum 1:10 in sterile saline solution to obtain the desired concentration of 10⁶ cfu/ml.

12.  Pipette 0.1 ml of the 10⁶ cfu/ml inoculum and transfer to a well, sterile test tubes of the same size, may be used to hold the diluted standardized inoculum.

13.  Inoculate plates with 10 µl.

14.  When inoculating manually, it is only important to include a drug free or control plate at the beginning of the inoculation series.

15.  Inoculate the bacterial suspensions onto the surface of the agar plate.

INCUBATION

16.   Incubate the plates in an inverted position at 30°C for 18-24 hours.

17.  Read and record the MIC at the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the naked eye.

18.  Report result as Resistant (R), Intermediate (I) or Susceptible (S).

Example: Antibiotic: Oxytetracycline MIC breakpoint: 0.2 µg/ml Interpretation: susceptible.

Stock Solution Preparation

a. At least 1,000 µg/ml or 10 times the highest concentration to be tested is to be prepared as an antimicrobial agent stock solution.

Some antimicrobial agents are of limited solubility. Therefore, lower concentration may be required.

 b. Some drugs must be dissolved in solvents other than water.

Prepare stock solutions using formula

1000  xVxC=W    P

 

  

P-potency (µg/mg), V-Volume required (ml), C-Final concentration of solution (mg/L), W-Wt of antibiotics dissolved in volume V (mL).