1. Susceptibility (Sensitivity) Testing: Results Pharmaceutical Microbiology – Practical Course Semester One_2014-2015 Sensitivity Results & MIC Broth Dilution/2014-2015 1

2. 2 Comment: E. coli is sensitive to Amikacin, resistant to Gentamicin and has intermediate sensitivity to Novobiocin and Penicillin. Measure diameter (in mm) of the zones of inhibition and compare to diameters in antibiotic table.

3. Determination of Minimum Inhibitory Concentration (MIC) by Broth Dilution Method Pharmaceutical Microbiology – Practical Course Semester One_2014-2015 Sensitivity Results & MIC Broth Dilution/2014-2015 3

4. Definition  It is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. 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. Sensitivity Results & MIC Broth Dilution/2014-2015 4 Minimum Inhibitory Concentration: Definition

5. Common Methods used for MIC Determination MIC determination by Broth Dilution Method MIC determination by Agar Diffusion Method Sensitivity Results & MIC Broth Dilution/2014-2015 5

6. MIC Determination by Broth Dilution Method (Serial Dilution Method) Principle  Add double strength broth media in all tubes  Serial dilution of the selected antibiotic  Inoculation with the isolated pathogenic microorganism  Incubation for 24 hour  Observation of the turbidity as a parameter of growth Sensitivity Results & MIC Broth Dilution/2014-2015 6

7. Detailed procedure Steps: 1.Label the test tubes (# 1-6 and C) 2.Aseptically distribute 1 ml double strength nutrient broth into each wasserman tube with a 5 ml pipette. 3.Add 1 ml antibiotic into the first wasserman tube. Mix and transfer 1 ml from tube # 1 to tube # 2. Repeat till tube # 6 using 1 ml pipette. Remove 1 ml from tube #6 after mixing and empty it in the sink. 4.Inoculate all tubes with 0.5 ml of the microorganism using a new 1 ml pipette. 5.Place in incubator at 37°C overnight and inspect for turbidity of media in test tubes. Sensitivity Results & MIC Broth Dilution/2014-2015 7 C Antibiotic

8. C Calculations Antibiotic Sample Test Tube Test Tube #1 Test Tube #2 Test Tube #3 Test Tube #4 Test Tube #5 Test Tube #6 Control Test Tube Concentration after addition of AB (mg/ml) 200100502512.56.253.125- Final Concentration (mg/ml) after addition of microorganism -66.733.316.78.34.172.083- Sensitivity Results & MIC Broth Dilution/2014-2015 8

9. Precautions Mark carefully all tubes (#1-6 and C) Adjust the volume in all tubes Don’t open the broth tube more than 2 times !!DO NOT ADD AB TO THE CONTROL TUBE!!  Mixing should be at the bottom of the tube? To avoid air bubbles ?? Contamination !!!!!!!! YOU MUST MUST MUST AVOID ANY CONTAMINATION Sensitivity Results & MIC Broth Dilution/2014-2015 9

10. 10 Expected Results Minimum Inhibition Concentration is equal to the concentration of the antibiotic in test tube #4 = 8.3 ug/ml (Case #1) 12 3 4 5 6C

11. Other Expected Results Sensitivity Results & MIC Broth Dilution/2014-2015 11 All tubes are CLEAR (CASE #2) Comment: MIC is less than concentration of the antibiotic in tube # 6 (i.e. < 2.083 ug /ml) All tubes are TURBID (CASE #3) Comment: MIC is higher than concentration of the antibiotic in tube # 1 (i.e. > 66.7 ug/ml) 1 2 3 4 5 6 C 1 2 3 4 5 6 C