1. PHT 381 Lab # 7

2. Antibiotic Sensitivity Testing Antibiotic sensitivity testing is used to determine the susceptibility of the microorganism to various antimicrobial agents.

3. Sensitivity testing techniques: Sensitivity testing techniques: Disc Diffusion Method.Disc Diffusion Method. Serial Dilution Method (Minimum inhibitory concentration).Serial Dilution Method (Minimum inhibitory concentration).

5. Disc Diffusion Method  The effectiveness of an antibiotic in this technique is based on the size of the zone of inhibition that surrounds a disc that has been impregnated with a specific concentration of the agent.  Advantages: Rapid Rapid Accurate Accurate Inexpensive Inexpensive

6. The recommended medium → Mueller Hinton agar ** The pH of the medium ( 7.2-7.4) **5% defibrinated sheep blood is added to the medium for certain fastidious organisms.

7. The inoculum: The turbidity of a broth culture / saline suspension of the test organism has to match a defined standard “0.5 McFarland” (a barium sulphate standard) ( the matched inoculum should give confluent growth).

8. Types of antibiotic:- Types of antibiotic:- Narrow spectrum antibiotic active against either Gram +ve bacteria only or gram –ve bacteria onlyNarrow spectrum antibiotic active against either Gram +ve bacteria only or gram –ve bacteria only Broad spectrum antibiotic active against both Gram’s +ve and -ve bacteriaBroad spectrum antibiotic active against both Gram’s +ve and -ve bacteria

10. Disc Diffusion method Materials: Materials: Cultures ofCultures of Staph. aureus, Staph. aureus, E. coli E. coli Pseudomonas aeruginosa Pseudomonas aeruginosa Filter paper discFilter paper disc Antibiotic solutions (Vancomycin, Augmentin, Ceftazidime, Azactam)Antibiotic solutions (Vancomycin, Augmentin, Ceftazidime, Azactam) 20 ml melted nutrient agar20 ml melted nutrient agar Petri-dishPetri-dish Sterile 1ml pipetteSterile 1ml pipette

11. Procedure: 45°c 0.1ml m.o inoculate Aug AzVan Cef Az 24 h at 37°c incubate Don’t invert

12. Results: Measure the diameter of each inhibition zone Measure the diameter of each inhibition zone * The diameter of the inhibition zones are directly proportional to the susceptibility of the microorganism to the antibiotics. Cef Az Van Aug

13. Results: Results: Mo MoAB S E Ps Ps 1 2 3 4