MICROBIOLOGY LABORATORY 5 Єtest Susceptibility Testing

USTMED ’07 Sec C – AsM; Photos provided by JV.N. & MeaM.

Procedure
SENSITIVITY TESTING 1. inoculation of the
organism for testing
Antibiotic Sensitivity Testing Using The Kirby-Bauer
using cotton swab
Procedure
by overlapping
streaking using
Kirby Bauer method. The test, introduced by William Kirby and
Mueller Hinton Agar
Alfred Bauer in 1966, consists of exposing a newly-seeded lawn
Plate.
of the bacterium to be tested, growing on a nutrient medium
(Mueller-Hinton agar) to filter paper disks impregnated with
2. Overlaying of Єtest
various antibiotics. The culture is incubated for 16 to 18 hours
strip on the
and then examined for growth. If the organism is inhibited by
previously
one of the antibiotics, there will be a zone of inhibition around
inoculated culture
the disk, representing the area in which the organism was
media
inhibited by that antibiotic.
3. Reading of the strip and recording the results
The diameter of the zone of inhibition around an antibiotic disk
is an indication of the sensitivity of the tested microorganism
to that antibiotic. The diameter of the zone, however, is also
related to the rate of diffusion of the antibiotic in the medium.
This fact must be kept in mind then interpreting the zone of
inhibition of various antibiotics.

MATERIALS:
- culture of the organisms in Mueller-Hinton agar plate
with antibiotic sensitivity discs
- Ruler graduated in millimeters

NOTE:
- The complete procedure is in you(r) lab [lab u 2!
Disk Diffusion Method
Hahaha!] manual.
Procedure
1. Measure the zone of inhibition and record your
1. dip the sterile swab into bacterial suspension
results.
compared to 0.5 MF standard then swab onto the
2. Interpret the results based on the table provided
surface of Mueller Hinton Agar using Overlapping
technique.
2. Allow the organism to be absorbed by the medium.
Place the appropriate antimicrobial (sensitivity) discs
using the dispenser or a sterile forceps. Incubate for
24 hours at 37o
3. Reading and Interpretation of the results. Measure
the diameter of the Zone of Inhibition (area wherein
there is no growth around the discs) using the
millimeter of a ruler. Record your results and
Interpret based on the table provided. Determine if
the Antibiotic (organism) is Sensitive, Intermediate or
Resistant. If there is overlapping in the zone of
inhibition, you can just measure the radius and
multiply the reading by 2 to get the diameter.

E. coli ATCC 25922. The

isolate tested on this Mueller-
Hinton agar plate is
interpreted as susceptible (S)
Єtest – The Problem Solver in Antimicrobial to all antimicrobial agents.
Susceptibility Testing Reading clockwise from the
top, MZ, AN, AM, CZ, CTX,
Etest is an antimicrobial CXM, CF, GM, NN: the three
gradient strip for the discs in the center of the
quantitative determination plate are SXT, FOX and TIM.
of susceptibility or
resistance of Pseudomonas aeruginosa, a
microorganisms. It is a resistant strain. Growth on
robust and simple this Mueller-Hinton agar plate
technique, minimally indicates that the isolate is
affected by laboratory resistant to six of 12
variations and can be used antimicrobial agents and
to test most susceptible to the remaing.
microorganisms. An The isolate is resistant to
accurate and reproducible SXT, GM, ATM, TIM, TIC and
Minimum Inhibitory MMZ. The isolate is
Concentration (MIC) is susceptible to CIP, AN, NN,
generated for reliable CA, IPM and PIP.
guidance of antimicrobial
therapy. ustmedc3@yahoogroups.com
audrey_cl@yahoo.com
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