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).
