PHT 416 Lab no 10 Minimum Inhibitory Concentration [MIC]

MBC(minimum bactericidal conc.): 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 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: o.1 ml S Incubate all tubes at 37°c for 24h 1ml reject 1ml AB MO 1ml 3 1 2 4 5 6 7 AB DSNB -ve Control +ve Control o.1 ml S Incubate all tubes at 37°c for 24h

Determination of MIC by the serial dilution method If Original Antibiotic Conc. = 80 µg/ml Result: Tube# 1 2 3 4 5 6 7 AB conc - Turbidity 40 20 10 5 2.5 1.25 -ve -ve -ve +ve +ve +ve +ve No bacteria No AB conc of the last tube clear + conc of first tube turbid 2 MIC =

Sensitivity Testing

Sensitivity Testing Sensitivity testing is used to determine the susceptibility of the microorganism 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.

“0.5 McFarland” (a barium sulphate standard) 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: incubate Don’t invert 0.2ml inoculate 45°c Aug Cef 24 h m.o 0.2ml Az inoculate 45°c Aug Cef incubate 24 h at 37°c Az Van Don’t invert

Results: 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. Van Aug Az Cef

Sensitivity Testing Results: ( in mm) Mo AB E 1 2 3 4

Thank you