-: Antimicrobial Susceptibility Testing and Antimicrobial Resistance:Antimicrobial Agent – Any chemical or biological agent that either destroys or inhibits the growth of microorganisms.
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Aim: - i) The main of in vitro antimicrobial susceptibility testing is to find out the susceptibility of a given
microorganism to known concentrations of the durg
ii) The test is routinely performed in microbiology laboratoty for determination of susceptibility of clinical
isolates.
iii) Two types of susceptibility testing methods are used:
1. Diffusion tests – commonly used
2. Dilution tests
Diffusion Tests
Disc diffusion method is most commonly used.
Disc Diffusion Method
Principle : The placing of a filter paper disc ( measuring 6 mm in diameter containing known amount of an
antimicrobial agent on the agar surface preciously inoculated with bacterium to be tested will result in zone of
inhibition of growth around the disc.
Following two methods are used:
1. Kirby-Bauer disc diffusion method
2. Stoke’s disc diffusion method.
1. Kirby-Bauer Disc Diffusion Method
It is a simple and reliable method applicable in routine clinical bacteriology
Requirements.
Disc of antimicrobial agents: Disc of a standard filter paper charged with appropriate concentration of the
antimicrobial agents.
Culture medium: Which supports the growth of most of the organisms? Following media are used,
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Mueller-Hinton agar
Nutrient agar
Blood agar/Chocolate agar – for fastidious bacteria, which do not grow on
nutrient agar.
Inoculum of test bacterium in a suitable broth medium ( e.g. peptone water). For preparation of inoculum,
pure culture of the organism is inoculated into a broth medium and incubated at 37° C for 2-4 hous.
Procedure.
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A standardize inoculums is inoculated with the help of a sterile cotton swab on the surface of the agar plate
Disc of antimicrobial agents are placed on the surface of agar plate
The plates are incubated at 37°C for 16-18 hours and susceptibility is determined on the basis of zone of
inhibition
A standard control strain is also tested for comparison
INTERPRETATION: The diameter of the zoned of growth inhibition around each disc are measured and
compared with zones of inhibition of standard control strain and results are interpreted as:
Sensitive: When zone diameter of test organism is greater than, equal to or not more than 4 mm less than
that of control strain.
Moderately sensitive: If its zone diameter is at least 12 mm but reduced by more than 4 mm as compared to
control strain.
Resistant: If it shown no zone of inhibition of growth or if the zone diameter is not more than 10 mm.
2. Stoke’s Disc Diffusion Method.
This method is similar to Kirby-Bauer disc diffusion method. The only change employed in this method is that
the control culture and test culture are grown on the same plate to compare the zones of inhibition produced in two
cultures by the same disc.
Procedure.
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The test bacterium is inoculated on middle third of culture plate and control culture on upper and
lower third of the plate. An uninoculated strip of about 5 mm between the test and control areas is
left for placing of discs. After inoculation, discs are placed (maximum -06), three on each side
between the control and test inocula.
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The plates are incubated at 37°C for 16-18 hours and observed for zones of inhibition.
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The results are interpreted by measuring and comparing the zones of inhibition of control and test
bacterium.
Dilution Tests – Here, serial dilutions of the drug are prepared and inoculated with the test bacterium.
Broth Dilution Test
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Serial dilutions of antimicrobial agents are incorporated into Mueller-Hinton broth in test tubes.
These media are then inoculated with test bacterium
Control strain with known susceptibility is also inoculated in a separate set
Incubated at 37°C for 16-18 hours
Results.
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The end points taken as MIC or MBC
MIC (Minimum inhibitory concentration) is the amount of antimicrobial agent required to inhibit growth.
MBC (Minimum bactericidal concentration) is the amount of antimicrobial agent required to kill the bacteria.
The MIC is detected by noting the lowest concentration of the antimicrobial agent showing no visible
growth.
For detection of MBC, a loop full form each tube is inoculated over different sector of plate and plates are
incubated at 37°C for 16-18 hours and the tube containing the lowest concentration of the antimicrobial
agent that fails show growth on subculture is taken MIC
 Dilution test are generally employed when the therapeutic dose is to be regulated accurately as in the
treatment of bacterial endocarditis, for tests
growing bacteria such as tuberelis.
Antibiotic assay in body fluid:
These are required to verify whether adequate drugs concentration are achieved in blood and other body fluids to
guard against excessive blood level of potential toxic drugs. The assays are generally done by making serial dilution
of the specimen and inoculating standard suspensions of bacteria of known MIC. Assays can also be done by the agar
diffusion method. This depends on the direct relationship between antibiotic concentration and the diameter of the
zone of inhibition with a standard sensitive strain of bacterium.