Antimicrobial_Sensitivity_Testing_Lecture.pptx

Antimicrobial_Sensitivity_Testing_Lecture.pptx

1. Antimicrobial Sensitivity Testing in Medical Microbiology Emphasis on Disk Diffusion (Kirby–Bauer) and MIC Methods Prepared for Lecture Presentation

2. Introduction Antimicrobial Sensitivity Testing (AST) determines bacterial response to antibiotics. Purpose: - Guides clinicians in antibiotic selection. - Monitors resistance patterns. - Supports infection control and antibiotic stewardship.

3. Principles of AST Bacteria classified as: - Susceptible (S) - Intermediate (I) - Resistant (R) Based on: - Minimum Inhibitory Concentration (MIC) - Zone of Inhibition Standards by CLSI and EUCAST define breakpoints.

4. Methods of AST 1. Diffusion Methods: - Disk Diffusion (Kirby–Bauer) - E-test (Gradient diffusion) 2. Dilution Methods: - Broth dilution (Macro/Micro) - Agar dilution 3. Automated Systems (VITEK, MicroScan)

5. Disk Diffusion Method (Kirby–Bauer) - Principle • Antibiotic diffuses radially from a disk into inoculated agar. • Bacteria inhibited where antibiotic concentration exceeds MIC. • Zone of inhibition measured in millimeters. • Compared to CLSI/EUCAST charts for S/I/R categorization.

6. Disk Diffusion - Procedure 1. Prepare 0.5 McFarland bacterial suspension. 2. Swab evenly on Mueller–Hinton agar plate. 3. Place antibiotic disks 24 mm apart. 4. Incubate at 37°C for 16–18 hours. 5. Measure zone diameters and interpret results.

7. Disk Diffusion - Example Chart | Antibiotic | Zone Diameter (mm) | Interpretation | |-------------|--------------------|----------------| | Ampicillin | ≥17 | Susceptible | | | 14–16 | Intermediate | | | ≤13 | Resistant |

8. MIC (Minimum Inhibitory Concentration) Definition: Lowest antibiotic concentration that inhibits visible bacterial growth. Methods: - Broth Dilution - Agar Dilution - E-test (Gradient Diffusion) MIC gives quantitative results useful for dose optimization.

9. Broth Dilution Method • Prepare serial twofold dilutions of antibiotic in broth. • Inoculate standardized bacterial suspension. • Incubate 18–24 h at 37°C. • Lowest concentration without visible growth = MIC.

10. E-Test Method • Gradient strip containing antibiotic concentrations placed on inoculated agar. • After incubation, the intersection of the elliptical zone and strip = MIC value. • Combines simplicity of diffusion with quantitative MIC accuracy.

11. Comparison: Disk Diffusion vs MIC | Feature | Disk Diffusion | MIC (Dilution) | |----------|----------------|----------------| | Output | Zone diameter | MIC value (µg/mL) | | Type | Qualitative | Quantitative | | Cost | Low | High | | Use | Routine | Precise dosing & resistance analysis |

12. Quality Control in AST • Use ATCC control strains (E. coli 25922, S. aureus 29213). • Maintain proper media pH, depth, and incubation conditions. • Use updated CLSI/EUCAST breakpoints. • Store disks at 2–8°C, protect from moisture and light.

13. Clinical Significance • Ensures effective antimicrobial therapy. • Prevents emergence of multidrug-resistant bacteria. • Supports hospital antibiotic stewardship programs. • Guides infection control measures.

14. Summary • AST is vital for guiding therapy and preventing resistance. • Kirby–Bauer: simple, qualitative method for routine testing. • MIC methods: quantitative, precise, and useful for dose decisions. • Both are complementary in modern diagnostic microbiology.