TABLE 7.
5: Classification of antimicrobial susceptibility testing
methods
.Diskdiffusion methods:
.Kirby-Bauer disk diffusion method
Stokes disk diffusion method
Dilution tests:
Broth dilution method
.Agardilution method
E-test
Automated methods
Molecular methods (PCR detecting drug resistant genes)
first-line antibiotics testeu
Diffusion Method
Kirby-Bauer Disk
into inoculum and squeezed to
A cotton swab is dipped
Then the swab is inoculated on
drain out the excess fluid.
the swab
to the Mueller-Hinton agar plate by streaking
three times over the entire agar surface.
After drying the surface of agar plate for 3-5 minutesthe
antibiotic disks are applied using either sterile forceps
or multidisk dispenser.
Disks should not be placed closer than 20 mm (center
to center) on the MHA plate.
maximum up to 6 disks can be applied on
Ordinarily,
in the and one in the
a 100 mm
plate (five periphery
center) (Fig. 7.2).
The plates are
then incubated at 37°C for 16-18 hours.
When tested for MRSA, result should be read only
after
24 hours of incubation.
around
The zones of complete growth of inhibition
Vernier
each of the disks are measured using a ruler or
The diameter of the disk is also included in this
caliper.
measurement. (Fig. 7.3)
zone
size into sensitive,
The interpretation of
zone
intermediate or resistant is based on the standard
size interpretation chart (Table 7.6).
a new
Control strains should be tested each time when
batch of disks or Mueller-Hinton agar is used.
Zone of inhibition
to
(diameter from edge
edge)
No zone surrounding
as
the disk reported
6 mm
Stokes Disk Diffusion Method
Here, the MHA plate is divided into three
parts. The test
organism is inoculated on the central one third and the
control strain on upper and lower thirds of the
In modified Stokes disk
plate.
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 37°C for 16-18 hours.
Reporting in Stokes Method
The sensitivity report is prepared by comparing the zones of
inhibition of control and test bacterium (Fig. 7.4). The radius
of the inhibition zone from the edge of the disk to the edge of
the zone is measured. Result is interpreted as follows:
Sensitive (S): Zone radius is wider than or equal to, or
not more than 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.
Dilution Tests
each
antimicrobial agent is serially diluted,
Here, the
with the test organism for antimicrobial
dilution is tested
and the MIC is calculated.
susceptibility test
concentration) is the lowest
MIC (minimum inhibitory
antimicrobial agent that will inhibit
concentration of an
a microorganism after overnight
the visible growth of
incubation.
whether dilutions of the
the
Depending upon
antimicrobial agent are made in agar or broth,
there are
two tests.
types of dilution
Broth Dilution Method
Serial dilutions of the antimicrobial agent in Mueller-
Hinton broth are taken in tubes and each tube is
inoculated with a fixed amount of suspension of the test
organism. A control organism ofknown sensitivity should
also be tested. Tubes are incubated at 37°C for 18 hours.
The MICis determined bynotingthelowest concentration
of the drug at which there is no visible growth, i.e. broth
appears clear. (Fig. 7.5).
The minimum bactericidal concentration (MBC) can be
obtained by subculturing fromn each tube (showing no
growth) onto a nutrient agar plate without any antimi-
crobial agent. The tube containing the lówest concentra-
tion of the drug that fails to show
is the MBC of the
growth, on subculture,
drug for that test strain (Fig. 7.5).
Broth dilution test can also be
done using microtiter
plates, such method is called as micro dilution method.
Agar Dilution Method
Here, the serial dilutions of the drug areprepared in
molten agar and poured into Petri dishes. The test strain
is spot inoculated. This method is more convenient than
broth dilution and has the added advantage of:
Several strains can to be tested at the same time by
using the same plate
It directly measures the MBC; there is no need of sub-
culturing as it is done with broth dilution method.
MEM
M I C = 0.6 ug/m
Fig.7.6: Epsilometer or E-test
Epsilometer or E-test
This is a quantitative method of detecting MIC by using the prin-
ciples of both dilution and diffusion of antibiotic into the medium
It uses an absorbent strip containing predefined gradient
(serial dilution) of antibiotic concentration immobilized
along its length.
It is applied to a lawn inoculum of a bacterium. Following
incubation of the test organism, an elliptical zone ofinhibition
is produced surrounding the strip.
The antibiotic concentration at which the ellipse edge
intersects the strip, is taken as MIC value (Fig. 7.6).
Automated Antimicrobial Susceptibility Tests
Several automated systems are available now, such as:
VITEK 2 bacterial identification and antimicrobial sensitivity
system (bioMerieux)
Phoenix System (Becton Dickinson)
Micro Scan Walk Away systemm
Most systems are computer assisted and have sophisticae
rates and determinethe antibio
SOftwares to analyzethe growth available panels thdt
Susceptibility report. They use commercially eby
contain added growth factors to speed organism growth, thet ds.
traditional methou
providing more rapid results compared with
Molecular Methods r'esistant
PCR based assays are available targeting specific drug res
genes; for example mecA gene for MRSA detection.
