Disk Diffusion Susceptibility Testing (Kirby-Bauer
I have been asked many times to interpret the susceptibility
results from labs outside ADDL. Even though the majority of
labs are following the correct protocol, some of you still
don't have the current guidelines. The following rules are
summarized from documents issued by The National Committee
on Clinical Laboratory Standards for susceptibility test.
I hope you will find this helpful in evaluating your procedures.
Please keep in mind that these rules are for non-fastidious
- Isolated colonies of each organism that might be playing
a pathogenic role should be selected from primary agar plates
and tested for susceptibility. Identification procedures
may be performed at the same time. Mixtures of different
types of microorganisms should not be tested on the same
plate. The practice of conducting susceptibility tests directly
with clinical material should be avoided. When the nature
of the infection is not clear and the specimen contains
mixed growth or normal flora, in which the organisms probably
bear little relationship to the infection being treated,
susceptibility tests are often not necessary and the results
can be grossly misleading.
- Of the many media available, NCCLS recommends Mueller-Hinton
agar due to: it results in good batch-to-batch reproducibility;
it is low in sulfonamide,trimethoprim, and tetracycline
inhibitors; it results in satisfactory growth of most bacterial
pathogens; and a large amount of data has been collected
concerning susceptibility tests performed with this medium.
If batches of media do not support adequate growth of organism,
the zone size will be larger and provide false result. Thus,
only media from manufacturers following the NCCLS standards
are to be used.
- The agar medium should have pH 7.2 to 7.4 at room temperature.
The surface should be moist but without droplet of moisture.
The antibiotic disks should be maintained at 8°C or lower
or freeze at -14°C or below until needed, according to the
manufacturer's recommendations. Allow the disks to warm
to room temperature before use. Don't use expired disks.
- To standardize the inoculum density, a BaS04 turbidity
standard is used (0.5 McFarland standard,approx. 10s
organism per mL).
- The steps of the standard method are as follows:
- a. Select at least 4 to 5 well-isolated colonies of the
same morphological type from an agar plate. Touch the top
of each colony with a wire loop and transfer the growth
to a tube containing 4 to 5 mL of a suitable broth medium,
such as tryptic-soy broth. Allow the broth culture to incubate
at 35°C until it achieves or exceeds the turbidity of 0.5
McFarland standard. For routine susceptibility tests, however,
the inoculum can also be prepared by making a direct saline
or broth suspension of colonies that are selected from an
18 to 24-hour agar plate (a nutrient, non-selective agar
such as blood agar plate must be used).
- Adjust the turbidity with sterile saline or broth. Use
adequate light, and, to aid in the visual comparison, read
the tube against a white background with contrasting
- Within 15 minutes after adjusting the turbidity of the
inoculum suspension, dip a sterile non-toxic swab on an
applicator into the adjusted suspension. Rotate the swab
several times, pressing firmly on the inside wall of the
tube above the fluid level to remove excess inoculum from
- Inoculate the dried surface of a Muller-Hintonagar plate
by streaking the swab over the entire sterile agar surface.
Repeat this procedure two more times, and rotate the plate
60° each time to ensure an even distribution of inoculum.
Replace the plate top and allow 3 to 5 minutes, but no longer
than 15 minutes, for any excess surface moisture to be absorbed
before applying the antibiotic disks. There should be
an almost confluent lawn of growth when done properly. If
only isolated colonies grow, the inoculum was too light
and the test should be repeated. To avoid extremes in inoculum
density, never use undiluted overnight broth cultures for
- Place the appropriate disks evenly (no closer than 24
mm from center to center) on the surface of the agar plate
either by using a sterile forceps or the dispensing apparatus.
No more than 12 disks should be placed on one 150 mm plate
or more than 5 disks on a 100 mm plate. A disk is not to
be moved once it has come in contact with the agar surface
since some of the compound diffuses almost instantaneously./li>
- Invert the plate and place them in an incubator at 35°C
within 15 minutes after disks are applied. The plates should
be incubated aerobically (no C02). After 16-18 hrs.of incubation,
examine each plate and measure the diameters of the zones
of complete inhibition, including the diameter of
the disk. Measure the zones to the nearest millimeter using
a ruler. Large colonies growing within a clear zone of inhibition
should be subcultured, reidentified and retested./li>
- Interpret the zone sizes by referring to the manufacturer
provided standard table and report the organism to be either
susceptible, intermediate, or resistant. Never compare the
zone sizes of two different antibiotics and judge their
- Quality control organisms such as E. coli ATCC 25922,
and S.aureusATCC 25923 should be tested periodically to
validate the accuracy of your procedures.
- prepared by ChingChingWu,DVM,PhD