The document discusses antibiotic sensitivity testing (AST), which measures the susceptibility of bacterial isolates to different antibiotics. AST aims to select the most appropriate antibiotic for patients and assess emerging resistance patterns. Common AST methods include disk diffusion, broth dilution, Etest, and automated systems. Disk diffusion is the most widely used method and involves placing antimicrobial disks on agar plated with the test organism and measuring inhibition zone sizes. Broth dilution determines the minimum inhibitory concentration (MIC) by visually inspecting bacterial growth in serial antibiotic dilutions.

1. ANTIBIOTIC SENSITIVITY TEST
(AST)

2. 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.

3. 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

4. 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.

5. 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

6. 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)

7. 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

8. 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.

9. 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.

10. • 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.

11. 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.

12. 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

13. 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.

14. 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.

15. The Kirby-Bauer Disc diffusion method
15
Transmitted
Light

17. 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.

18. Commonly used disk concentrations and interpretation
of disk diffusion test

19. 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.

20. 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.

21. 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.

23. STOKES’ METHOD

24. 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. BROTH DILUTION METHOD
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. 27
BROTH DILUTION METHOD
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

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

29. 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

30. The gradient technique, Etest

31. 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

32. 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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