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Antimicrobial Susceptibility Testing

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99 views

Antimicrobial Susceptibility Testing

Good for students

Uploaded by

maxwell amponsah
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Accra Technical University

Department of
Medical Laboratory Technology

Course Name: MICROBIOLOGY V


Course Code: MLM 413

TOPIC:
ANTIMICROBIAL SUSCEPTIBILITY TESTING

LECTURER: DR. HENRY HACKMAN


ANTIMICROBIAL SUSCEPTIBILITY TESTING

 The AST measures the ability of antibiotic to inhibit or kill


bacterial growth in vitro.
 guide to clinicians in selecting the appropriate antibiotic,
 epidemiological data,
 reduces the use of empirical therapy since empirical therapy
is effective for a few pathogens, notably Group A
streptococcus.
Methods of Antimicrobial Susceptibility Testing (AST)

 Diffusion Method (Kirby-Bauer Disc Diffusion Method, Agar


well diffusion method - Qualitative method)
 Dilution Method (Minimum Inhibition Concentration -
Quantitative method)
 Etest (AB Biodisk)
 Automation/Semi-automation (Mini-API, Vitek 2 Compact
System etc)
Diffusion Method:
Kirby-Bauer Disc Diffusion Method
 A standard bacterial suspension is prepared (0.5 McFarland).
 A sterile cotton swab is dipped into the standardized bacterial
suspension and uniformly spread on the surface of the
Muellar-Hinton agar plate.
 The agar surface is allowed to dry for about 5 minutes.
 Discs impregnated with a specified concentration of a
selected antimicrobial agent are placed on the surface of the
Mueller Hinton agar medium with sterile forceps.
 The plate is immediately placed in an incubator with plates
inverted.
Agar Well Diffusion Method

 A standard bacterial suspension is prepared (0.5 McFarland).


 A sterile cotton swab is dipped into the standardized bacterial
suspension and uniformly spread on the surface of the
Muellar-Hinton agar plate.
 The agar surface is allowed to dry for about 5 minutes.
 Wells of 6mm diameter are generated in the agar using sterile
cork borer. The wells are filled with 10µl of the antimicrobial
agent (specified concentration).
 The plate is immediately placed in an incubator with plates
inverted.
The Incubating Conditions

 Temperature: 35-370C
 Duration: 16 - 18hrs
 Environment: 02
 N. meningitis, H. influenzae and S. pneumoniae, extra CO2
 For enterococci and vancomycin or teicoplanin, incubate for
24hrs
 Incubate plates within 15mins of application of discs
 During incubation the various drugs diffuse radially outward
through the agar producing an antibiotic concentration
gradient.
Interpretation of Zones of Inhibitions

 Clear zone of inhibition (diameter including the disc) are


observed and measured after incubation. Resistant strains
may have no zones of inhibition
 The diameter of the zone of inhibition is measured with a
metric ruler to the nearest mm.
 The wider the zone of inhibition, the more susceptible the
pathogen is.

Interpretation of Zones of Inhibitions

 Results are interpreted using a breakpoints (concentration of


antibiotics) that relate zone diameter to the degree of
microbial resistance or susceptibility.
 The values in the table (chart) are derived by finding the MIC
values and zone diameters for many microbial strains by
international standardized institutions such as Clinical &
Laboratory Standards Institute (CLSI) and European
Committee on Antimicrobial Susceptibility Testing (EUCAST).
 Results are qualitative - sensitive, intermediate / indeterminate
(not reported) or resistant
Dilution Method

 Quantitative estimation of antimicrobial activity


 Minimal Inhibitory Concentration (MIC): The lowest
concentration of a specific antimicrobial drug preventing
growth of a particular pathogen.
 Minimal Lethal Concentration (MLC): the lowest drug
concentration that kills the pathogen.
 Serial dilution (2 – fold dilution)
 Gold standard for determining susceptibility of organisms
 There are 2 types: Tube/Broth and Agar Dilution
Tube/Broth Dilution Method

 Muellar Hinton broth or Nutrient broth are prepared and


poured into two sets of test tube series.
 Serial dilutions generating decreasing concentrations of the
antimicrobial agent are prepared in one set of the tubes
containing a suitable growth medium (Mueller Hinton broth,
nutrient broth).
 A known volume (10µl) of the inoculum (suspension of
organism with 0.5 McF) is added to the two set of tubes
containing the broth.
Tube/Broth Dilution Method

 Microbial growth in the other set of tubes without the


antimicrobial agent will be used as the control tubes.
 The tubes are incubated for 16 – 18hrs at 37°C and then are
examined for visible growth or turbidity by determining the
optical density at 620nm.
 The lowest concentration of drug that prevents growth of the
microorganism is the MIC
 MLC is determined by assaying for live organisms in those
tubes from the MIC test that showed no growth.
 Antimicrobial agent concentration in the blood is important to
determine the drug MIC to be used to treat an infection.
Tube/Broth Dilution Method
Agar Dilution Method

 Plates containing Mueller- Hinton agar are spread with specific


standard bacteria suspension.
 Various concentrations (mg/ml) of the antimicrobial agents are
prepared.
 Filter papers are dipped into each concentration and placed on the
agar plate (agar disk diffusion) and examined for growth. OR
 Wells are generated in the agar and same volume of varied
concentration of antimicrobial agent is poured into the well (agar well
diffusion) and examined for growth.
 The lowest concentration that prevents the microbial growth is the MIC.
E-TEST (AB Biodisk)

 The E-test from AB Biodisk may be used in susceptibility testing


particular for anaerobic pathogens.
 A petri dish of the agar is streaked in three different directions with
the test organism and special plastic Etest strips are placed on the
surface so that they extend out radially from the centre.
 Each strip contains a gradient of an antibiotic and is labeled with a
scale of minimal inhibitory concentration values. The lowest
concentration of the strip lies at the centre of the plate.
 After 24 – 48 hours of incubation, an elliptical zone of inhibition
appears. MICs are determined from the point of intersection
between the inhibition zone and the strips scale of MIC values.
E-TEST (AB Biodisk)
Automated or Semi-Automated Systems

 MINI-API (bioMérieux, France)


 A suspension of the test organism is prepared and inoculated
into the wells of Mini API test strips using the appropriate
reagents according to manufacturer’s instruction.
 The strips are incubated and read on the MINI-API strips
reader to determine the organism and its sensitivity profile to
specific antibiotics.
VITEK 2 Compact System (bioMérieux, France)

 Rapid automated microbiological system


 Uses an advanced expert system.
 Gram staining of bacterial colonies
 Inoculum preparation and incubation in the Vitek
 Bacteria and yeast identification to the species level using 60
biochemical tests
 Antimicrobial susceptibility testing (AST)
 Analyzes MIC patterns of several antimicrobials
VITEK 2 Compact System (bioMérieux, France)

 Interprets MIC as susceptible, intermediate and resistant


 Detects resistance mechanism detection
 Epidemiologic trending and reporting

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