Antimicrobial Susceptibility

Test

Serial Dilution Method (Minimum

inhibitory concentration MIC).
Introduction
◼ Susceptibility test, main purposes:
◼ As a guide for treatment
◼ Sensitivity of a given m.o. to known conc. of
drugs
◼ Drug concentration in body fluids or tissues( must
be above the level of MIC )

◼ As an epidemiological tool
The emergence of resistant strains of major
pathogens (e. g. Shigellae, Salmonella typhi)
◼ As a parameter for antibiotics activity in
the research
Antimicrobial Susceptibility Testing

◼ Broth dilution method

This test used for determining levels of microbial
resistance to an antimicrobial agent where a
serial dilutions of the test agent are made in a
liquid microbial growth medium, which is
inoculated with a standardized number of
organisms and incubated for a prescribed time
where the lowest concentration of the agent in
the serial dilution preventing appearance of
turbidity (growth ) is considered to be the
(MIC)
Dilution Method
◼ Broth dilution method
◼ Permit quantitative results:
◼ Indicating amount of a given drug necessary
to inhibit (bacteriostatic activity) or kill
(bactericidal activity) the microorganisms
tested

◼ Minimum Inhibition Concentration (MIC)

◼ Minimum Bactericidal Concentration (MBC)
Dilution Method
◼ Minimum Inhibition Concentration (MIC)
◼ The lowest concentration of antimicrobial
agent that inhibits bacterial growth/
multiplication

◼ Minimum Bactericidal Concentration (MBC)

or Minimum Lethal Concentration (MLC)
◼ The lowest concentration of antimicrobial
agent that allows less than 0.1% of the
original inoculum to survive
Broth Dilution Method
◼ Procedure
◼ Prepare bacterial solution adjusted with
MacFarland NO. 0.5
◼ Prepare a stock solution of antimicrobial agent
◼ Find the break point of that antimicrobial agent
on this M.O. from references
◼ Prepare serial dilutions of that antimicrobial
agent by using sterilized broth (like BHI broth)
in a concentrations above and below the break
points.
◼ Inculcate 1 ml of bacterial solution in each
tube of the serial dilution
◼ Prepare two controls (no antibiotics, no test
bacteria )
◼ Incubate the tubes at 37 C for 18 hrs
◼ Turbidity visualization → MIC
◼ Subculturing of non-turbid tubes, overnight
◼ Growth → MBC
 Broth Dilution Method

Record visual turbidity

64 32 16 8 4 2 1 C1 C2
Subculture non-turbid tubes
to agar plates (use 0.01 ml
standard loop)
MIC = 16 mg/l
0.01 ml (spread on plate), Incubate 37 oC,
o/n
MBC = 32 mg/l

Controls:
C1 = No antibiotic
64 32 16
C2 = No test bacteria
Broth Dilution Method
◼ Disadvantages :
◼ Only one antibiotic & one organism can be
tested each time
◼ Time-consuming
◼Thank you