PHT 416PHT 416 Lab no 10Lab no 10

MIC: It is the lowest concentration

of the antimicrobial agent that inhibits the growth of the test organism but not necessarily kills it.

MBC(minimum bactericidal conc.):

It is the lowest concentration of the antimicrobial agent that kills the test organism.

Bactericidal drugs have MBC 2-4 times MIC.

Bacteriostatic drugs have MBC much higher than MIC.

The breakpoint of an antimicrobial agent:

It is the concentration of the drug which can be achieved in the serum with optimal dose.

Susceptible organisms: are those with MICs at/below the breakpoint.

Resistant organisms: are those with MICs above the breakpoint.

INDICATIONS FOR DETERMINING THE MIC

1 -Serious infections e.g., infective endocarditis.

2 -Slow-growing organisms e.g., Mycobacterium tuberculosis.

METHODS FOR DETERMINATION OF MIC

1 .Serial dilution method

2 .Agar diffusion method

3 .E- test

DETERMINATION OF MIC BY THE SERIAL DILUTION METHOD

Materials: Staph. aureus Cefoxitin 80µg ⁄ml Double strength nutrient broth (DSNB) 7 sterile test tubes 1 sterile pipette 5 ml 2 sterile pipette 1 ml

Procedure:

DSNB

1ml1 2 3 4 5 6 7

AB

1ml

1ml 1ml 1ml 1ml 1ml

1ml reject

S

o.1

ml

ABMO

-ve Control +ve Control

Incubate all tubes at 37°c for 24h

DETERMINATION OF MIC BY THE SERIAL DILUTION METHOD

Result:Tube#1234567

AB conc-

Turbidity

40 20 10 5 2.5 1.25

No AB

-ve -ve -ve +ve +ve +ve +ve

No bacteria

If Original Antibiotic Conc. = 80 µg/ml

conc of the last tube clear + conc of first tube turbidconc of the last tube clear + conc of first tube turbid

2 2 MIC MIC ==

SENSITIVITY TESTING

SENSITIVITY TESTING

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

It can be done by : Plate diffusion method using disc. Minimum inhibitory concentration [MIC].

SENSITIVITY TESTING

Antibiotic sensitivity testing by the disc diffusion method:

Principle; Rapid Accurate Inexpensive

The diameter of the resulting zones of inhibition that surrounds a disc that has been impregnated with a specific concentration of the agent will be a measure of the effectiveness of the antibiotics on the microorganism.

SENSITIVITY TESTING

There are two types of antibiotic:-

Narrow spectrum active against Gram +ve only or Gram –ve

only

Broad spectrum antibiotic active against both types of bacteria

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).

SENSITIVITY TESTING

Materials: Cultures of E. coli. Filter paper disc impregnated in antibiotic

solutions. Different antibiotic solutions. Muller Hinton agar plate. Loop. Forceps.

Procedure:

45°c

0.2ml

m.o

inoculate Aug

Az Van

Cef

Az

24 h

at 37°c

incubate

Don’t invertDon’t invert

RESULTS: Measure the diameter of each inhibition zoneMeasure the diameter of each inhibition zone

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

Cef

Az

VanAug

SENSITIVITY TESTING

Results: ( in mm)

MoMo

ABAB EE

11

22

33

4 4

THANK YOU