Kirby-Bauer antibiotic testing is a test which uses antibiotic-impregnated wafers to test
whether bacteria are affected by antibiotics. In this test, wafers containing antibiotics
are placed on an agar plate where bacteria have been placed, and the plate is left to
incubate. If an antibiotic stops the bacteria from growing or kills the bacteria, there
will be an area around the wafer where the bacteria have not grown enough to be visible.
This is called a zone of inhibition. The size of this zone depends on how effective
the antibiotic is at stopping the growth of the bacterium. A stronger antibiotic will
create a larger zone, because a lower concentration of the antibiotic is enough to stop growth.
The bacteria in question is swabbed uniformly across a culture plate. A filter-paper disk,
impregnated with the compound to be tested, is then placed on the surface of the agar.
The compound diffuses from the filter paper into the agar. The concentration of the compound
will be highest next to the disk, and will decrease as distance from the disk increases.
If the compound is effective against bacteria at a certain concentration, no colonies will
grow where the concentration in the agar is greater than or equal to the effective concentration.
This is the zone of inhibition. This along with the rate of antibiotic diffusion are
used to estimate the bacteria's sensitivity to that particular antibiotic. In general,
larger zones correlate with smaller minimum inhibitory concentration of antibiotic for
that bacteria. Inhibition produced by the test is compared with that produced by known
concentration of a reference compound. This information can be used to choose appropriate
antibiotics to combat a particular infection.
Standardization
Preparation All aspects of the Kirby-Bauer procedure are
standardized to ensure consistent and accurate results. Because of this, a laboratory must
adhere to these standards. The media used in Kirby-Bauer testing must be Mueller-Hinton
agar at only 4 mm deep, poured into either 100m or 150mm Petri dishes. The pH level of
the agar must be between 7.2 and 7.4. Inoculation is made with a broth culture diluted
to match a 0.5 McFarland turbidity standard, which is roughly equivalent to 150 million
cells per mL. Incubation Procedure
Using an aseptic technique, place a sterile swab into the broth culture of a specific
organism and then gently remove the excess liquid by gently pressing or rotating the
swab against the inside of the tube. Using the swab, streak the Mueller-Hinton
agar plate to form a bacterial lawn. To obtain uniform growth, streak the plate
with the swab in one direction, rotate the plate 90° and streak the plate again in that
direction. Repeat this rotation 3 times.
Allow the plate to dry for approximately 5 minutes.
Use an Antibiotic Disc Dispenser to dispense discs containing specific antibiotics onto
the plate. Using a flame-sterilized forceps, gently press
each disc to the agar to ensure that the disc is attached to the agar.
Plates should be incubated overnight at an incubation temperature of 37 °C.
Oxford Penicillin Cup Method Disks containing increasing antibiotic concentrations
are placed on seeded bacterial lawn on the agar surface and paltes are incubated. Zone
sizes are measured from the edge of the disk to the end of the clear zone. Interpretation
is more complicated in mixed susceptibility populations. These are plotted as linear dimensions
or squares of distances as a function of the natural logarithm of antibiotic concentration
in the disks. The MIC is determined from the zero intercept of a linear regression fit
through the data. The intercept itself is the logarithm of the MIC. The slope of the
regression line is related to the diffusion coefficient of that particular antibiotic
in the agar References