Hamburg 2 The conventional method of sensitivity testing is by the disc sensitivity test or agar dilution method. In the correct hands this is a powerful technique that is capable of showing the relative sensitivities of a range of antibiotics to a specific pathogen. The size of the zone of sensitivity is dependent on the MIC and the concentration of the interface between growth and no growth is the MIC.
•Laboratory studies on antibiotic
Dr. G. Kattam Maiyoh
01/23/15 GKM/KISIIU/MBSM713 /BIOC.AMIC.AGENS.LEC05 1
MBSM 713: BIOCHEMISTRY OF ANTI
Laboratory studies on antibiotic action
• In the laboratory, susceptibility is most often
measured using a disk diffusion test.
• Antibiotic solutions of particular concentrations
are dried onto filter paper disks.
• These are then applied to a lawn of the microbe
under examination which has previously been
inoculated onto an appropriate solid medium.
• Susceptible or resistant to antibiotic
• MIC = Minimum inhibitory concentration
• MBC = Minimum bactericidal concentration
• Minimum concentration required to inhibit growth
– Disc diffusion
– Agar dilution
– Breakpoint MIC
Diffusion of antibiotic from a paper disc
periphery of zone
equals the MIC
• Breakpoint: concentration above which the isolate is
described at resistant and below which is susceptible
e.g. S < 8mg/L R ≥ 8mg/L
Breakpoint = 8mg/L
• Range: Lowest to highest MIC for population
• MIC50Median for series of MICs
– MICs of population ordered from lowest to highest
– MIC value of the strains that appears 90% up the series.
– Antibiotic considered to be successful if > 90% of population inhibited.
– Also show if resistance is emerging i.e. 10% of population resistant.
Minimum Inhibitory Concentrations
MIC50, MIC90 and Range
Etest Determination of MIC
Breakpoint Test to Determine Bacterial
Inoculation of Plates
growth at fixed
shows resistanceAmoxycillin (4mg/L)
Gentamicin (2mg/L)Ciprofloxacin (1mg/L) Cefotaxime (2mg/L) Ceftazidime (4mg/L)
Control - No drug
Evaluation of Laboratory Tests
• MIC test on plates is the best
– Time consuming and costly
– Most detailed
• Disc test/Etest is easiest
– Requires more skill to interpret
– Least skill required
– Technique must be exact
– Can be read by computer
– Large amounts of data
Minimum Bactericidal Concentration
64 32 16 8 4 2 1 mg/l
Subculture onto drug-free agar
16 8 4 2 1 mg/l
Stokes controlled sensitivity test
• A control organism is inoculated on part of a
plate and the test organism is plated on the
• Disks are placed at the interface and the zones
of inhibition are compared.
• The use of a sensitive control shows that the
antibiotic is active, so that if the test organism
grows up to the disk it may safely be assumed
that the test organism is resistant to that
Stokes' sensitivity test
•The bacterium in the
diagram is susceptible to
drug "x" but resistant to
• The disc containing
drug "y" contains active
antibiotic as shown by the
zone of inhibition it
causes in the control
MIC and MBC
• An alternative measure of susceptibility is to
determine the Minimum Inhibitory
Concentration (MIC) and the Minimum
Bactericidal Concentration (MBC) of a drug.
• A series of broths are mixed with serially diluted
antibiotic solutions and a standard inoculum is
• After incubation, the MIC is the first broth in
which growth of the organism has been inhibited.
• The more resistant an organism is, then the
higher will be the MIC.
Minimum Bactericidal Concentration (MBC)
• The MBC is measured by inoculating the
broths used for MIC determinations onto
• The MBC is the first dilution at which no
growth is observed.
• Cidal drugs have MBC values that are close to
the MIC value for particular organisms.
• With static agents, the MIC is much lower
than the MBC.