Ast

ANTIBIOTIC SENSITIVITY TEST
(AST)

AIMS
To measure susceptibility of an isolate to range
of antibiotics.
To select the most appropriate antibiotic for
patients.
To assess emerging bacterial resistance
patterns.

Clinical & Laboratory Standards Institute
(CLSI)
Develop standards, methods, QC
parameters, and interpretive criteria for
sensitivity testing
If necessary, can alter the breakpoints
based on emerging resistance

Definition
• Minimum Inhibition Concentration (MIC)
The lowest concentration of antimicrobial agent
that inhibits visible growth/ multiplication of a
microorganism.
• Minimum bactericidal concentration (MBC)
The MBC is the lowest concentration of the
antibiotic that will kill a bacterial strain.

CHOICE OF ANTIBIOTIC
Follow CLSI recommendations
Each laboratory should have a battery of
antibiotics ordinarily used for testing
Use the least toxic, most cost-effective, and
most clinically appropriate agents
Refrain from more costly, broader-spectrum
agents
Selection of antimicrobial is based on the type
of organism being tested and source of the
isolate

METHODS
 Disk diffusion method
• Kirby Bauer disk diffusion method
• Stoke’s method
 Broth macrodilution / Tube dilution
 Broth microdilution
 Agar dilution method
 Gradient diffusion method (E-Test)
 Automated methods (VITEK)
 Molecular methods (PCR detecting drug resistant
genes)

DISK DIFFUSION METHODS
• Put a filter disc containing measured quantity
of drugs on a solid medium that has been
seeded with test bacteria
• Two types -
1. Kirby Bauer disk diffusion method
2. Stoke’s method

DISK DIFFUSION METHODS
• Disk diffusion tests are the most widely used
method.
• They are suitable for rapidly growing
pathogenic bacteria
• They are unsuitable for slow growing bacteria.

PRINCIPLE
• Antimicrobial disk placed on agar media.
• Antimicrobial begins to diffuse into surrounding
agar.
• A logarithmic reduction in concentration occurs
as the distance from the disk increases.
• The rate of diffusion of antimicrobial through the
agar is dependent upon
the diffusion and solubility properties of the drug in
agar and
the molecular weight of the antimicrobial compound.

• The antimicrobial inhibits the growth of
bacteria.
• The zone size of inhibition of growth is
influenced by
the depth of the agar medium
pH
presence of divalent cations
inoculum density and
temperature of incubation.

REQUIREMENTS
• Sterile liquid medium (peptone water broth) in
2ml tubes
• 0.5 McFarland standard and Wickerham card
• Mueller-Hinton agar plates
• Caliper or ruler
• Antibiotic disks
• Forceps, Antibiotic disk dispenser (optional)
• 18 to 24 hour old pure culture of the organism to
be tested
• Vortex
• Sterile swabs/Inoculating loop
• 35°C to 37°C non-CO2 incubator.

PROCEDURE- Kirby-Bauer Disc diffusion method
• Preparation of inoculum
Using a sterile inoculating loop, touch 4 or 5 isolated
colonies of the organism to be tested.
Suspend the organism in 2 ml of suitable liquid
medium (peptone water broth)
Vortex the broth tube to create a smooth suspension.
Adjust the turbidity of this suspension to
a 0.5 McFarland standard (1.5 x 108 cfu/mL)
• by adding more organism if the suspension is too light or
• diluting with sterile saline if the suspension is too heavy.
Use this suspension within 15 minutes of preparation

Inoculation of the MH plate
 Dip a sterile swab into the inoculum
tube.
 Rotate the swab against the side of
the tube (above the fluid level)
using firm pressure, to remove
excess fluid.
 Inoculate the dried surface of a MH
agar plate by streaking the swab
three times over the entire agar
surface; rotate the plate
approximately 60 degrees each time
to ensure an even distribution of
the inoculum.
 Rim the plate with the swab to pick
up any excess liquid
 Dry the plate for least 3 to 5
minutes, but no more than 15
minutes.

Placement of the antibiotic disks
 Place the appropriate
antimicrobial impregnated
disks on the surface of the
agar, using forceps to
dispense each antimicrobial
disk one at a time.
 When all disks are in place,
replace the lid, invert the
plates, and place them in a
35°C air incubator for 16 to
18 hours.

The Kirby-Bauer Disc diffusion method
15
Transmitted
Light

Interpretation and Reporting
• By comparing with the diameters with “standard
tables” - zone size is determined.
• Based on standard chart zone size is
susceptible (S), intermediate (I), or resistant (R).
 Susceptible ‘S’ - Interpretive category that indicates
an organism is inhibited by the recommended dose,
at the infection site.
 Intermediate “I” - Interpretive category that
represents an organism that may require a higher
dose of antibiotic for a longer period of time to be
inhibited
 Resistant “R” - Interpretive category that indicates an
organism is not inhibited by the recommended dose,
at the infection site.

Commonly used disk concentrations and interpretation
of disk diffusion test

STOKES METHOD
• The comparative disk test
• Uses both a test organism and a control
organism on the same plate.
• The control organism is of defined sensitivity
to the antibiotics being tested.
• Allows a direct comparison of the diameter of
the zones of inhibition between the test and
control organisms.

STOKES METHOD
• The MHA plate is divided into 3 parts.
• Test organism is inoculated on the central one
third and control strain on upper and lower thirds
of the plate.
In modified Stokes disk diffusion method- the test
bacterium is inoculated over the upper and lower
thirds of the plate and control on central one third.
• An Uninoculated gap of 2-3 mm wide should
separate, the test and the control area on which
the antibiotic disks are applied.
• The plates are then incubated at 370C for 16-18
hours.

Reporting in Stokes Method
• The sensitivity report is prepared by comparing
the zones of inhibition of control and test
bacterium.
• The radius of the inhibition zone from the edge of
the disk to the edge of the zone is measured.
Result is interpreted as:
• Sensitive (S): Zone radius is wider than or equal
to, or not >3 mm smaller than the control.
• Intermediate ( I): Zone radius is >2 mm but
smaller than the control by >3 mm.
• Resistant (R): No zone of inhibition or zone radius
measures 2 mm or less.

BROTH DILUTION METHOD
• Procedure
• Making serial dilutions (2-fold) of antibiotic in
broth Mueller-Hinton, Tryptic Soy Broth
Inoculation of fix bacterial inoculum, incubate
overnight
Controls:
• no inoculum
• no antibiotic
Turbidity visualization  MIC
MBC can be obtained by subculturing from each tube
to a nutrient agar plate without any antimicrobial
agent

26
Day 1
Add 1 ml of test bacteria
(1*106 CFU/ml) to tubes
containing 1 ml broth and
concentration of
antibiotic (mg/l)
Controls:
C1 = No antibiotic, check
viability on agar plates
immediately
C2 = No test bacteria
Bacterial conc.= 5*105 CFU/ml
Incubate 35 oC, o/n
128 64 32 16 8 4 2 C1 C2
64 32 16 8 4 2 1 C1 C2

27
Day 2
Record visual turbidity
Subculture non-turbid tubes
to agar plates (use 0.01 ml
standard loop)
MIC = 16 mg/l
64 32 16 8 4 2 1 C1 C2
0.01 ml (spread plate), Incubate 35 oC, o/n
64 32 16
Day 3
Determine CFU on plates:
At 16 mg/ = 700 CFU/ml >
0.1% of 5*105 CFU/ml
MBC = 32 mg/l

Antimicrobial susceptibility testing using micro-broth
dilutions
•
•
•
•
•
•
•
• •
•
•
•
•
96 well microtiter plate
ug/ml
64 32 16 8 4 2

Epsilometer /E test
• This is a quantitative method of detecting MIC by
using the principles of both dilution and diffusion
of antibiotic into the medium
• The Etest technique comprises a predefined
gradient (serial dilution) of antibiotic
concentrations on a plastic absorbent strip.
• applied to a lawn inoculum of a bacterium.
following incubation of the test organism, an
elliptical zone of inhibition is produced surrounding
the strip
• The antibiotic conc. at which the ellipse edge
intersect the strip is taken as MIC value.
29

AUTOMATED SYSTEMS
• Systems are computer assisted and have
sophisticated software to analyse the growth rate
and determine the antibiotic susceptibility report.
• Detect growth in microvolumes of broth with various
dilutions of antimicrobials
• Detection via photometric, turbidimetric, or fluorometric
methods
• Types
BD Phoenix system (Becton Dickinson)
Vitek 1 and 2 (bioMerieLLx)
Micro Scan Walk Away system
TREK Sensititre

AUTOMATED SYSTEMS
• Advantages
Increased reproducibility
Decreased labor costs
Rapid results
Software
• Detects multi-drug resistances
• ESBLs
• Correlates bacterial ID with sensitivity
• Disadvantages
– Cost

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