Antimicrobial Sensitivity Testing

 A test done to check the

effectiveness of a drug against a
bacterium and to select the best
drug that acts against the
bacterium.
Antibiotic Sensitivity Test
 The in vitro testing of bacterial cultures
with antibiotics to determine
susceptibility of bacteria to antibiotic
therapy.
 Antibiotic Sensitivity Tests

Diffusion &
Diffusion Dilution
Dilution

Kirby-Bauer Stokes Tube Agar

E-Test
Method Method Dilution Dilution

Qualitative Methods Quantitative Methods

Diffusion methods
 Disk Diffusion Method
 Principle
– A paper disk with a defined amount of antibiotic is
used to generate a dynamically changing gradient
of antibiotic concentrations in the agar in the
vicinity of the disk.
 The basics
 The antibiotic contained in a reservoir is
allowed to diffuse out into the medium and
interact in a plate freshly seeded with the test
organisms.
 The disk is applied to the surface of an agar
plate inoculated with the test organism.
– The antibiotic diffuses out of the disk to form the
gradient.
– The test organism starts to divide and grow and
progresses toward a critical mass of cells.
 Inhibition zone edge is formed at the critical
time where a particular concentration of the
antibiotic is just able to inhibit the organism
before it reaches an overwhelming cell mass
or critical mass.
INHIBITION ZONE
EDGE
Kirby-Bauer Method
 ANTIBIOTIC SENSITIVITY
TESTS
Kirby–Bauer method
 Most used
 Zones of inhibition around the discs
are recorded and interpreted
 Guidelines issued by
- Clinical and Laboratory
Standards Institute (CLSI)
- European Committee on
Antimicrobial Susceptibility Testing
(EUCAST)
Fig. 11.4 Zones of inhibition around antibiotic discs on the lawn culture of the
test bacterium: (a) sensitive strain (b) resistant strain (Source: Department of
Microbiology, Pondicherry Institute of Medical Sciences, Puducherry) and (c)
schematic diagram of the Kirby–Bauer disc diffusion test (Source:
https://commons.wikimedia.org/wiki/File:Zones_of_Inhibition.png)
MATERIALS

Mueller-
Hinton Agar

Antibiotic
Disks

Turbidity
Standard

Swabs
 Mueller-Hinton Agar
 Medium containing beef
infusion, peptone, and
starch.
 Used primarily for the disk-
diffusion method.

Robust red algae (Solieria robusta)

Source of Agar
 Mueller-Hinton agar is considered the BEST
for routine susceptibility testing of nonfastidious
bacteria.

Why?
 It shows acceptable batch-to-batch
reproducibility for susceptibility testing.
 It is low in sulfonamides, trimethoprim, and
tetracycline inhibitors.
 It gives satisfactory growth of most
nonfastidious pathogens.
 Mueller-Hinton Agar
 Cool the medium to 45–50 ⁰C and pour into
the plates. Allow to set on a level surface, to a
depth of approximately 4 mm.
– A 9-cm plate requires approximately 25 ml of
medium.
 When the agar has solidified, dry the plates for
10–30 minutes at 35 ⁰C by placing them in the
upright position in the incubator with the lids
tilted.
– If it is not to be used immediately, the agar medium
can be stored in a refrigerator (2 to 8C) for 2 weeks.
MATERIALS

Mueller-
Hinton Agar

Antibiotic
Disks

Turbidity
Standard

Swabs
 Antibiotic Disks
 Any commercially available discs with the
proper diameter (6 mm) and potency can be
used.
 Stocks of antibiotic discs can be stored at
-20 ⁰C for 1 month.
– On removal from the refrigerator, the containers
should be left at room temperature for about 1
hour to allow the temperature to equilibrate.
Antibiotic Disks
MATERIALS

Mueller-
Hinton Agar

Antibiotic
Disks

Turbidity
Standard

Swabs
 Turbidity Standard
 0.5 McFarland
 Prepared by pouring 0.6 ml of
a 1% (10 g/l) solution of
barium chloride dihydrate
into a 100-ml graduated
cylinder, and filling to 100ml
with 1% (10 ml/l) sulfuric
acid.
MATERIALS

Mueller-
Hinton Agar

Antibiotic
Disks

Turbidity
Standard

Swabs
 Cotton Swabs
 A supply of cotton wool swabs on wooden
applicator sticks should be prepared.
 They can be sterilized in tins, culture tubes, or
on paper, either in the autoclave or by dry
heat.
Cotton Swabs
 Procedure

Application of Antibiotic Discs

Incubation
At 35⁰C for 16-18 hours

Measurement of inhibition zone diameter

 Kirby-Bauer Method

Procedure
1.Inoculum: To prepare the inoculum from
the primary culture plate, touch with a loop
the tops of each of 3–5 colonies, of similar
appearance, of the organism to be tested.
2. Transfer this growth to a tube of saline.
3. Compare the
tube with the
turbidity standard
and adjust the
density of the test
suspension to that
of the standard by
adding more
bacteria or more
sterile saline.
4. Inoculate the plates by dipping a sterile
swab into the inoculum.
 Remove excess inoculum by pressing and rotating the
swab firmly against the side of the tube above the
level of the liquid.
5. Streak the swab all over the surface of the
medium three times, rotating the plate
through an angle of 60⁰ after each application.

6. Finally, pass the swab round the edge of the

agar surface.
7. Leave the inoculum to dry for a few minutes
at room temperature with the lid closed.
8. The antibiotic discs may be placed on the
inoculated plates using
– sterile forceps.
– a template.
– a sterile needle tip.
– antibiotic disc dispenser.
 Application of Antibiotic Discs

Incubation
At 35⁰C for 16-18 hours

Measurement of inhibition zone diameter

!
 A maximum of seven discs can be placed
on a 9–10 cm plate.
– Six discs may be spaced evenly, approximately
15 mm from the edge of the plate, and 1 disc
placed in the centre of the plate.
 The plates should be placed in an
incubator within 30 minutes of
preparation.
 Temperatures above 35⁰C invalidate
results for oxacillin/methicillin.
 Do not incubate in an atmosphere of
carbon dioxide.
 Disks should not be moved after diffusion.
 Application of Antibiotic Discs

Incubation
At 35⁰C for 16-18 hours

Measurement of inhibition zone diameter

Measurement of diameter
 Using a ruler
– on the under-surface
of the plate containing
transparent medium.
 Using a pair of
calipers
– on the plate
containing opaque
medium.
Measurement of diameter
 Using automated zone readers
– BIOMIC
– Aura
– Protozone
 ANTIBIOTIC SENSITIVITY TESTS

Fig. 11.7 MIC of colistin by E-test for

Pseudomonas aeruginosa—the ellipsoidal zone
Fig. 11.6 Stokes method of testing
for antibiotic susceptibility of inhibition has a cut-off where the zone meets

the strip incorporated with gradient

concentration of the antibiotic (MIC is 1.5
μg/mL) (Source: Department of Microbiology,

Pondicherry Institute of Medical Sciences,

Puducherry)
 STOKES METHOD

• PLATE IS DIVIDED INTO THREE PARTS. TEST ORGANISM

INOCULATED AT CENTRAL 1/3rd AND CONTROL ON UPPER
AND LOWER THIRD. MAXIMUM OF 6 DISCS CAN BE
INOCULATED ON SINGLE PLATE.
• MODIFIED STOKES METHOD
 REPORTING
 SENSITIVE: ZOI greater than or not more than
7 mm smaller than that of control.
 INTERMEDIATE: ZOI more than 2mm but is
smaller than that of the control by more than
7mm.
 RESISTANT : ZOI <2mm
Interpretation of results
 Using a ruler
 The diameter of the
zone of inhibition is
measured using a
ruler or a pair of
calipers.
– This diameter is
interpreted according
to the critical
diameters.
 Interpretative chart of zone sizes
Diameter of zone inhibition (mm)
Antibiotic
Resistant Intermediate Susceptible

Tetracycline <14 15-18 >19

Chloramphenicol <12 13-17 >18

Cotrimoxazole <10 11-15 ≥16

Nitrofurantoin <14 15-16 >17

Erythromycin <13 14-22 >23

Gentamycin <12 13-14 >15

 Factors influencing
size of zone
 Inoculum density
– Too light inoculum
• Inhibition zones will be larger even though the
sensitivity of the organism is unchanged
Relatively resistant strains may be falsely reported as
susceptible.
– Too heavy inoculum
• Inhibition zones will be smaller
Relatively susceptible strains may then be falsely
reported as resistant.
• Timing of disk application
Plates seeded with test strain

Left at room temperature for long

periods
Multiplication of inoculum before
disks are applied

Reduction in zone diameter

Susceptible strain reported as

resistant
 Temperature of incubation
– If the temperature is lowered, the time required
for effective growth is extended and larger
zones result.
 Potency of antibiotic disks
– If the potency of the drug is reduced owing to
deterioration during storage, the inhibition
zone will show a corresponding reduction in
size.
Dilution methods
 Dilution methods
 Used to determine the minimal concentration
of antibiotic to inhibit or kill the
microorganism.
 Achieved by dilution of antibiotic in either
agar or broth media.
 Tube Agar
dilution dilution

MIC
Minimum inhibitory concentration
 The lowest concentration of drug that
inhibits the growth of the bacteria
isolated from the patient.
 The MIC is determined by inoculating the
organism isolated from the patient into a
series of tubes or cups containing progressive
dilutions of the drug.
 Tube dilution
Patient's organism is added to tubes
containing decreasing amounts of the
antibiotic

Incubation
At 37°C overnight

Lowest concentration of drug that

inhibits growth is the MIC
MIC
Minimum bactericidal concentration

 The lowest concentration of drug that kills

the bacteria isolated from the patient.
 Agar dilution
 Serial dilutions of the drug are prepared in
agar and poured into plates.
 Advantage
– Many strains can be inoculated on each plate
containing an antibiotic dilution.
 Broth microdilution
 Broth microdilution plate contains
– Each row:
• standard dilutions of eight bacterial organisms in each row (denoted by
letters A-H).
– Each column
• contains a standard antibiotic concentration that doubles when moving from
right to left.
 The minimum inhibitory concentration (MIC) is determined by the
first well where there is no visible growth.
E-Test
 What is E-Test?
 Epsilometer Test
 Quantitative method of antibiotic sensitivity
testing.
 Applies both dilution of antibiotic and
diffusion of antibiotic into the medium.
 Combines the principles of disk diffusion and
agar dilution methods

Diffusion

E-Test
Dilution
 A predefined stable antibiotic gradient is
present on a thin inert carrier strip.
 Using innovative dry chemistry technology,
E-Test is used to determine the on-scale
Minimum Inhibitory Concentration (MIC).
 E-Test
Procedure
Apply E-Test strip on an inoculated agar plate

Immediate release of drug

Incubation of plate

Symmetrical inhibition ellipse is produced

The intersection of the inhibitory zone edge and the
calibrated carrier strip indicates the MIC with inherent
precision and accuracy.
MIC
AST of Fastidious organisms
 Most fastidious organisms do not grow well
enough in routine antibiotic testing systems
and require some type of supplementation.

Pathogen Medium
Streptococcus pneumoniae Mueller-Hinton sheep blood agar

Haemophilus sp. Haemophilus Test Medium (Mueller-

Hinton Agar, β NAD, bovine hematin,
yeast extract)

Neisseria gonorrheae Thayer-Martin agar

AST can be done with automation
 Automated Susceptibility Methods:

 Determination of bacterial growth in wells containing

antimicrobial agent are performed in short period of time
using computer-assisted instruments.
Various techniques include:

 Turbidimetric detection: VITEK (report ready in 4-15 hours)

 Flourimetric detection: AutoSCAN Walkaway (report ready in

3.5-7 hours)

 Video Image processing: ALADIN (report ready in 18-24 hours)

 ANTIBIOTIC
SENSITIVITY
TESTS
AUTOMATED
METHODS TO DETECT
ANTIBIOTIC
SUSCEPTIBILITY
i) Automated
microtitre plates
ii) VITEK system

Fig. 11.9 VITEK system for rapid

identification of bacteria and
determining the MIC levels of a
set of antibiotics for an organism
(Source: Centre for Virology and
Molecular Biology, Pushpagiri
Institute of Medical Sciences and
Research Centre, Thiruvalla,
Kerala)