PHT 381PHT 381

Lab # 7Lab # 7

Antibiotic Sensitivity Antibiotic Sensitivity TestingTesting

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

Sensitivity testing Sensitivity testing techniques:techniques:

•Disc Diffusion Method.Disc Diffusion Method.

•Serial Dilution Method Serial Dilution Method (Minimum inhibitory (Minimum inhibitory concentration).concentration).

Disc Diffusion MethodDisc Diffusion Method The effectiveness of an antibioticThe effectiveness of an antibiotic in in

this technique is based on this technique is based on the size of the size of the zone of inhibitionthe zone of inhibition that surrounds a that surrounds a disc that has been impregnated with a disc that has been impregnated with a specific concentration of the agent.specific concentration of the agent.

Advantages:Advantages:Rapid Rapid Accurate Accurate Inexpensive Inexpensive

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.

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).

Types of antibiotic:-Types of antibiotic:-

• Narrow spectrum antibioticNarrow spectrum antibiotic active against either Gram +ve active against either Gram +ve bacteria only or gram –ve bacteria onlybacteria only or gram –ve bacteria only

• Broad spectrum antibioticBroad spectrum antibiotic active against both Gram’s +ve and active against both Gram’s +ve and -ve bacteria-ve bacteria

Disc Diffusion methodDisc Diffusion method Materials:Materials:

• Cultures Cultures of of Staph. aureus, Staph. aureus, E. coli E. coli Pseudomonas aeruginosaPseudomonas aeruginosa• Filter paper disc Filter paper disc • Antibiotic solutionsAntibiotic solutions (Vancomycin, (Vancomycin,

Augmentin, Ceftazidime, Azactam)Augmentin, Ceftazidime, Azactam)• 20 ml 20 ml melted nutrient agarmelted nutrient agar• Petri-dishPetri-dish• Sterile 1ml pipette Sterile 1ml pipette

Procedure:Procedure:

45°c

0.1ml

m.o

inoculate Aug

Az Van

Cef

Az

24 h

at 37°c

incubate

Don’t invertDon’t invert

Results:Results: Measure the diameter of each inhibition Measure the diameter of each inhibition

zonezone

* The diameter of the inhibition zones are * The diameter of the inhibition zones are directly proportional to the susceptibility directly proportional to the susceptibility of the microorganism to the antibiotics.of the microorganism to the antibiotics.

CefAz

VanAug

Results:Results:

MoMo

ABAB SS EE PsPs

11

22

33

44