Faculty of Medicine and Health Sciences Microbiology Lab Second semester 2013-2014

1. Faculty of Medicine and Health Sciences Microbiology Lab Secondsemester 2013-2014 prepared by: Mohammad Al-Qadi E-mail: m.qadi@najah.edu

2. Antibiotic Susceptibility testing (Broth dilution and disk diffusion procedures) Lab # 8

3. Introduction • Antimicrobial Susceptibility Testing Methods: The procedures used to detect antimicrobial resistance • MIC (minimum inhibitory concentration): The lowest concentration of the antibiotic capable of preventing growth of the test organism. • MBC (minimum bactericidal concentration): The lowest concentration of the chemotherapeutic agent that results in no growth (turbidity) of the subcultures.

4. Components of Antimicrobial Susceptibility Testing Bacterial inoculum size –McFarland Growth medium –Mueller-Hinton Incubation temperature Incubation atmosphere Incubation duration Antimicrobial concentrations tested

5. Methods that can be used to Measure Antimicrobial Activity • Conventional susceptibility testing methods (broth dilution , agar dilution & disk diffusion) • Commercial susceptibility testing systems. (antibiotic strips & computerized automated tests)

6. Conventional Testing Methods General considerations: • Inoculum preparation • Selection of antimicrobial agents for testing • Standardization: 1. Optimize bacterial growth conditions 2. Optimize conditions for maintaining antimicrobial integrity and activity 3. Maintain reproducibility & consistency

7. Procedure & PrincipleTube dilution tests • In this test, a series of culture tubes are prepared, each containing a liquid medium and a two-folds serial dilution of an antibiotic. • The tubes are then inoculated with the test bacterium and incubated for 16-20 hours at 37C. • After incubation, the tubes are examined for turbidity (growth). The lowest concentration of the antibiotic capable of preventing growth of the test organism is the minimum inhibitory concentration (MIC). • Subculturing of tubes showing no turbidity into tubes containing medium but no antibiotict can determine the minimum bactericidal concentration (MBC). MBC is the lowest concentration of the chemotherapeutic agent that results in no growth (turbidity) of the subcultures. • These tests, however, are rather time consuming and expensive to perform.

8. Disk Diffusion Advantages 1. Convenient 2. 12 antimicrobial agents can be tested for one bacteria 3. Results are accurate

9. Inoculum Preparation • Pure culture • Standardized inoculum ? -McFarland turbidity standards -0.5 McFarland = 1.5 x 108 CFU / ml -1 % sulfuric acid + 1.175 % barium chloride

10. Procedure & PrincipleThe disk diffusion test (Bauer-Kirby test) • Prepare a standard turbidity inoculumof the test bacterium so that a certain density of bacteria will be put on the plate. • Inoculate a 150mm Mueller-Hinton agar platewith the standardized inoculum so as to cover the entire agar surface with bacteria. • Place standardized antibiotic-containing discs on the plate. • Incubate the plate agar side up. incubate at 37°C for 16-18 hours. • Measure the diameter of any resulting zones of inhibition in millimeters (mm) . • Determine if the bacterium is susceptible, moderately susceptible, intermediate, or resistant to each antibiotic using a standardized table.

11. Result

12. Why McFarland standards

13. Why McFarland standards

14. Commercial Susceptibility Testing Systems

15. Commercial Susceptibility Testing Systems

16. Automated tests • Computerized automated tests have been developed for antimicrobial susceptibility testing. • These tests measure the inhibitory effect of the antimicrobial agents in a liquid medium by using light scattering to determine growth of the test organism. • Results can be obtained within a few hours. • Labs performing very large numbers of susceptibility tests frequently use the automated methods but the equipment is quite expensive.

17. End