Determination of MIC by Agar Diffusion Method 1252022

Determination of MIC by Agar Diffusion Method 1/25/2022 Pharmaceutical Microbiology. PM 704 1

Minimum Inhibitory Concentration (MIC) o Definition: is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. o WHY? Clinically, MIC is not only used to determine the amount of antibiotic that the patient will receive but also the type of antibiotic used. This will lower the opportunity for microbial resistance to specific antimicrobial agents. 1/25/2022 Pharmaceutical Microbiology. PM 704 2

Common methods used for MIC determination n MIC by broth dilution method n MIC by agar diffusion method 1/25/2022 Pharmaceutical Microbiology. PM 704 3

MIC by agar diffusion method o Principle n Different gradient of AB solution n Inoculation with the isolated pathogenic microorganism n Incubation for 24 hour n Observation of the diameter of the zone of inhibition as a parameter of the growth 1/25/2022 Pharmaceutical Microbiology. PM 704 4

Principle 1/25/2022 Pharmaceutical Microbiology. PM 704 5

Principle (Cont. ) 1/25/2022 Pharmaceutical Microbiology. PM 704 6

Detailed procedure o Materials: n n n n Nutrient agar tube Sterile Petri dish Test tube with bacterial culture 2 sterile Pasteur pipettes Sterile Cork borer Sterile blade 4 Wassermann tubes with 4 different concentrations of antibiotic 1/25/2022 Pharmaceutical Microbiology. PM 704 7

Detailed procedure 1/25/2022 Pharmaceutical Microbiology. PM 704 8

Precautions o Pour the agar at optimum temp. (< 50 o. C) o Mix very well o Flame the blade very well after and before each cup o Don’t leave any agar debris inside the cup o Transfer the antibiotic from the low to the high concentration o AVIOD THE OVERFLOW, only two drops in each cup. o !!DO NOT INVERT THE PLATE!! o Transfer the plate very carefully 1/25/2022 Pharmaceutical Microbiology. PM 704 9

Expected results 1/25/2022 Pharmaceutical Microbiology. PM 704 10

Results Antibiotic Concentration µg/ml Log concentration Zone Diameter (mm) 100 2 17 250 2. 39 20 1500 3. 17 26 3000 3. 47 29 1/25/2022 Pharmaceutical Microbiology. PM 704 11

Results Zone Diameter (mm) 20 18 16 14 12 10 8 6 4 2 0 0 0, 5 1 1, 5 2 Log MIC 1/25/2022 Pharmaceutical Microbiology. PM 704 2, 5 3 Log Conc 12

Results Zone Diameter (mm) 35 30 25 20 15 10 5 0 Log MIC 1/25/2022 0 0, 5 1 1, 5 2 Pharmaceutical Microbiology. PM 704 2, 5 3 Log Conc 13